Pan-tropic entry inhibitors

ABSTRACT

This disclosure relates to compounds according to Formula (I), salts, prodrugs and pharmaceutical formulation comprising the compound are provided herein for the treatment of CXCR4 and CCR5 related conditions. The conditions may include viral infections, abnormal cellular proliferation, retinal degeneration and inflammatory diseases, or the compounds may be used as immunostimulants or immunosuppressants. Furthermore, the compounds may be used in combination with another active ingredient selected from an antiviral agent or chemotherapeutic agent.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the National Stage of International Application No.PCT/US2019/023022, which claims the benefit of U.S. ProvisionalApplication No. 62/644,982 filed Mar. 19, 2018. The entirety of each ofthese applications is hereby incorporated by reference for all purposes.

TECHNICAL FIELD

The present disclosure relates to pan-tropic entry inhibitors, and inparticular, to chemokine CXCR4 and CCR5 receptor modulators and usesrelated thereto. In certain embodiments, the disclosure relates topharmaceutical compositions comprising compounds disclosed herein,derivatives, or pharmaceutically acceptable salts or prodrugs thereof.In certain embodiments, the compositions disclosed herein are used formanaging CXCR4 and CCR5 related conditions, typically prevention ortreatment of viral infections such as HIV or for managing cancer.

BACKGROUND

As of July 2017, approximately 21 million of the estimated 37 millionpeople worldwide living with HIV/AIDS have access to antiretroviraltherapy (ART). While this represents a significant improvement from2010, when only 7.5 million individuals had access to ART, it stillmeans that over 40% of HIV-positive individuals around the world remainwithout access to therapy. Moreover, in many parts of the world,economic considerations preclude the use of the more effective currentgeneration of HIV therapeutics, which are clearly superior to earlygeneration drugs. Thus, a need exists for new anti-HIV agents that can:(i) simplify dosing schedules, (ii) reduce pill burden and (iii) expandthe utility of existing drugs through new combinations thatsignificantly delay the onset of resistance. In vitro resistance studiesconducted with entry inhibitors TAK-779 (a CCR5 antagonist) and AMD3100required significantly more passages to develop resistance (41 and 63,respectively) when compared to the anti-HIV agents, AZT and nevirapine(11 and 2 passages, respectively), both of which target HIV reversetranscriptase (RT) (Balabanian et al., J Immun, 2004, 173(12); Cameronet al., PNAS USA, 2010, 107 (39); Mellors et al., Mol Pharma, 1992,41(3); Wu et al., PLoS Pathogens, 2009, 5(12)).

Gouwy et al. first demonstrated a synergistic effect with CCL5 andCXCL12 (the cognate ligands of CCR5 and CXCR4, respectively) againstviral replication in cell cultures infected with dual-tropic HIV. Thestudy also showed that a combination of the CXCR4 antagonist, AMD3100and the CCR5 antagonist, Maraviroc synergistically enhanced potencyagainst dual-tropic strains. Since the chemokine entry inhibitor,Maraviroc, only blocks entry of the M-tropic virus, its extended useleads to selection of the more virulent dual M/T-tropic and T-tropicstrains of the virus (Gouwy et al., Eur J Immun, 2011, 41(4); Rossettiet al., AIDS Res Human Retroviruses, 2014, 30(1)), followed by a rapiddecline in health. Administration of AMD11070 (a CXCR4 antagonist)results in a tropism shift in some mixed-tropic patients (Moyle et al.,Clin Inf Diseases, 2009, 48(6)) and long-term exposure in animals hasbeen linked to toxicity.

Studies have shown that CXCR4 interactions also regulate the migrationof metastatic cells. Hypoxia, a reduction in partial oxygen pressure, isa micro-environmental change that occurs in most solid tumors and is amajor inducer of tumor angiogenesis and therapeutic resistance. Hypoxiaincreases CXCR4 levels (Staller, et al. (2003) Nature 425: 307-311).Microarray analysis on a sub-population of cells from a bone metastaticmodel with elevated metastatic activity showed that one of the genesincreased in the metastatic phenotype was CXCR4. Furthermore,over-expression of CXCR4 in isolated cells significantly increased themetastatic activity (Kang, et al. (2003) Cancer Cell 3: 537-549). Insamples collected from various breast cancer patients, Muller et al.(Muller, et al. (2001) Nature 410: 50-56) found that CXCR4 expressionlevels are higher in primary tumors relative to normal mammary gland orepithelial cells. These results suggest that the expression of CXCR4 oncancer cell surfaces may direct the cancer cells to sites that expresshigh levels of SDF-1. Consistent with this hypothesis, SDF-1 is highlyexpressed in the most common destinations of breast cancer metastasisincluding lymph nodes, lung, liver, and bone marrow. Moreover, CXCR4antibody treatment has been shown to inhibit metastasis to regionallymph nodes when compared to control isotypes that all metastasized tolymph nodes and lungs.

In addition to regulating migration of cancer cells, CXCR4-SDF-1interactions may regulate vascularization necessary for metastasis.Blocking either CXCR4/SDF-1 interaction or the major G-protein ofCXCR4/SDF-1 signaling pathway (G_(αi)) inhibits VEGF-dependentneovascularization. These results indicate that SDF-1/CXCR4 controlsVEGF signaling systems that are regulators of endothelial cellmorphogenesis and angiogenesis. Numerous studies have shown that VEGFand MMPs actively contribute to cancer progression and metastasis.

Thus, there is a need to identify CXCR4 and/or CCR5 antagonists fortherapeutic applications in treating or preventing cancer and a need fornew entry inhibitors to alleviate some of the abovementioned problems,to some extent at least.

SUMMARY

According to a first embodiment of the present disclosure there isprovided a compound according to Formula (I) or salt thereof,

wherein R^(A), R^(B), R^(C) and R^(D) are individually and independentlyH, aryl or a C1 to C4 alkyl which may be straight, branched, saturatedor unsaturated, or R^(A) and R^(B), together with the atoms to whichthey are attached, or R^(C) and R^(D), together with the atoms to whichthey are attached, may be connected to form a carbocyle, heterocarbocylearyl or heteroaryl, and R^(A), R^(B), R^(C) and R^(D) may beindividually and independently optionally substituted with R; R^(E),R^(G1), R^(G2), R^(G3), R^(H1) and R^(H2) are each individually andindependently selected from an H, alkyl, carbocycle, heterocarbocyle,aryl or heteroaryl, each of which may be optionally substituted with Rx;Ring W is a carbocyle, heterocarbocyle aryl or heteroaryl which issubstituted with one or more RF groups, where o is 0, 1, 2, 3 or 4 orring W is absent; RF is a chloro, fluoro, bromo, iodo, C1 to C3 alkyl,trifluoromethyl, O; X is a N or a CH; Y is NH when Z is CO and Y is COwhen Z is NH or Y and Z are absent; n, m and p are each independently 0,1 or 2; s is 1, 2, 3, 4 or 5; and Rx is a halogen, nitro, cyano,hydroxy, trifluoromethoxy, trifluoromethyl, amino, formyl, carboxy,carbamoyl, mercapto, sulfamoyl, methyl, ethyl, methoxy, ethoxy, acetyl,acetoxy, methylamino, ethylamino, dimethylamino, diethylamino,N-methyl-N-ethylamino, acetylamino, N-methylcarbamoyl, N-ethylcarbamoyl,N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl,methylthio, ethylthio, methylsulfinyl, ethylsulfinyl, mesyl,ethylsulfonyl, methoxycarbonyl, ethoxycarbonyl, N-methylsulfamoyl,N-ethylsulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl,N-methyl-N-ethylsulfamoyl, carbocyclyl, aryl, or heterocyclyl.

Further features provide for the compounds of Formula (I) to be Formula(IA) or salts thereof,

wherein R^(A), R^(B), R^(C), R^(D), R^(E), R^(G1), R^(G2), R^(G3),R^(H1) and R^(H2), X, p, n and s are as defined above is an H, alkyl,carbocycle, heterocarbocyle, aryl or heteroaryl, each of which may beoptionally substituted with Rx.

Some exemplary compounds of Formula (I) are selected from the followinglist:

wherein R^(E) is an optionally substituted alkyl, carbocycle,heterocarbocycle, aryl, or heteroaryl such as:

In certain embodiments, the disclosure relates to isolated compositionscomprising compounds disclosed herein in substantially pure form.

In certain embodiments, the disclosure relates to a pharmaceuticalformulation comprising a compound as described herein including saltsand prodrugs thereof and a pharmaceutically acceptable excipient,diluent, or carrier. In certain embodiments, the pharmaceuticalcomposition is in the form of a tablet, pill, capsule, gel, or aqueousbuffered solution.

In certain embodiments, the disclosure relates to uses of compoundsdisclosed herein in the production of a medicament for the treatment ofCXCR4 and CCR5 related conditions, such as, viral infections, abnormalcellular proliferation, retinal degeneration, inflammatory diseases, oras an immunostimulant or immunosuppressant.

In certain embodiments, the disclosure relates to pharmaceuticalcompositions comprising a compound as described herein and anotheractive ingredient such as an antiviral agent or chemotherapeutic agent.

In certain embodiments, the disclosure relates to methods of treating orpreventing a viral infection, the method comprising administeringpharmaceutical composition comprising a compound as described hereinoptionally in combination with another active ingredient to a subject inneed thereof. In further embodiments, the subject is at risk of,exhibiting symptoms of, or diagnosed with a viral infection.

In certain embodiments, the disclosure relates to uses of a compound asdescribed herein in the production of a medicament for the treatment ofa viral infection. In a typically embodiment, the viral infection is anIV infection.

In certain embodiments, the disclosure relates to methods of treating orpreventing cancer, the method comprising administering a pharmaceuticalcomposition comprising a compound as described herein optionally incombination with another active ingredient to a subject in need thereof.In further embodiments, the subject is at risk of, exhibiting symptomsof, or diagnosed with cancer.

In certain embodiments, the disclosure relates to methods of makingcompounds disclosed herein comprising mixing starting materials andreagents under conditions such that the product is formed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of Maraviroc;

FIG. 2 shows the structure of compound 3 and a modeling picture in whichit is overlaid with Maraviroc;

FIG. 3 shows the structure of compound 4 and a modeling picture in whichit is overlaid with Maraviroc;

FIG. 4 is a graph showing the calculated IC₅₀ values of exemplarycompounds on P450 2D6.

FIG. 5 is a graph showing the calculated IC₅₀ values of exemplarycompounds on P450 3A4.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of medicine, organic chemistry, biochemistry,molecular biology, pharmacology, and the like, which are within theskill of the art. Such techniques are explained fully in the literature.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. In this specification andin the claims that follow, reference will be made to a number of termsthat shall be defined to have the following meanings unless a contraryintention is apparent.

Prior to describing the various embodiments, the following definitionsare provided and should be used unless otherwise indicated.

To the extent that structures provided herein are compounds withtautomers by hydrogen migration, a skilled artisan would understand theformula to cover all tautomeric forms.

As used herein, “alkyl” means a noncyclic straight chain or branched,unsaturated or saturated hydrocarbon such as those containing from 1 to10 carbon atoms, typically 1 to 4 otherwise designated C1-4alkyl.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, n-septyl, n-octyl, n-nonyl, andthe like; while saturated branched alkyls include isopropyl, sec-butyl,isobutyl, tert-butyl, isopentyl, and the like. Unsaturated alkylscontain at least one double or triple bond between adjacent carbon atoms(referred to as an “alkenyl” or “alkynyl”, respectively). Representativestraight chain and branched alkenyls include ethylenyl, propylenyl,1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl,3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and thelike; while representative straight chain and branched alkynyls includeacetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl,3-methyl-1-butynyl, and the like.

Non-aromatic mono or polycyclic alkyls are referred to herein as“carbocycles” or “carbocyclyl” groups. Representative saturatedcarbocycles include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,and the like; while unsaturated carbocycles include cyclopentenyl andcyclohexenyl, and the like.

“Heterocarbocycles” or “heterocarbocyclyl” groups are carbocycles whichcontain from 1 to 4 heteroatoms independently selected from nitrogen,oxygen and sulfur which may be saturated or unsaturated (but notaromatic), monocyclic or polycyclic, and wherein the nitrogen and sulfurheteroatoms may be optionally oxidized, and the nitrogen heteroatom maybe optionally quaternized. Heterocarbocycles include morpholinyl,pyrrolidinonyl, pyrrolidinyl, piperidinyl, hydantoinyl, valerolactamyl,oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydropyridinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl,tetrahydropyrimidinyl, and the like.

The term “aryl” refers to aromatic homocyclic (i.e., hydrocarbon) mono-,bi- or tricyclic ring-containing groups preferably having 6 to 12members such as phenyl, naphthyl and biphenyl. Phenyl is a preferredaryl group. The term “substituted aryl” refers to aryl groupssubstituted with one or more groups, preferably selected from alkyl,substituted alkyl, alkenyl (optionally substituted), aryl (optionallysubstituted), heterocyclo (optionally substituted), halo, hydroxy,alkoxy (optionally substituted), aryloxy (optionally substituted),alkanoyl (optionally substituted), aroyl, (optionally substituted),alkylester (optionally substituted), arylester (optionally substituted),cyano, nitro, amino, substituted amino, amido, lactam, urea, urethane,sulfonyl, and, the like, where optionally one or more pair ofsubstituents together with the atoms to which they are bonded form a 3to 7 member ring.

As used herein, “heteroaryl” or “heteroaromatic” refers an aromaticheterocarbocycle having 1 to 4 heteroatoms selected from nitrogen,oxygen and sulfur, and containing at least 1 carbon atom, including bothmono- and polycyclic ring systems. Polycyclic ring systems may, but arenot required to, contain one or more non-aromatic rings, as long as oneof the rings is aromatic. Representative heteroaryls are furyl,benzofuranyl, thiophenyl, benzothiophenyl, pyrrolyl, indolyl,isoindolyl, azaindolyl, pyridyl, quinolinyl, isoquinolinyl, oxazolyl,isooxazolyl, benzoxazolyl, pyrazolyl, imidazolyl, benzimidazolyl,thiazolyl, benzothiazolyl, isothiazolyl, pyridazinyl, pyrimidinyl,pyrazinyl, triazinyl, cinnolinyl, phthalazinyl, and quinazolinyl. It iscontemplated that the use of the term “heteroaryl” includes N-alkylatedderivatives such as a 1-methylimidazol-5-yl substituent.

As used herein, “heterocycle” or “heterocyclyl” refers to mono- andpolycyclic ring systems having 1 to 4 heteroatoms selected fromnitrogen, oxygen and sulfur, and containing at least 1 carbon atom. Themono- and polycyclic ring systems may be aromatic, non-aromatic ormixtures of aromatic and non-aromatic rings. Heterocycle includesheterocarbocycles, heteroaryls, and the like.

“Alkylthio” refers to an alkyl group as defined above attached through asulfur bridge. An example of an alkylthio is methylthio, (i.e., —S—CH₃).

“Alkoxy” refers to an alkyl group as defined above attached through anoxygen bridge.

Examples of alkoxy include, but are not limited to, methoxy, ethoxy,n-propoxy, i-propoxy, n-butoxy, s-butoxy, t-butoxy, n-pentoxy, ands-pentoxy. Preferred alkoxy groups are methoxy, ethoxy, n-propoxy,i-propoxy, n-butoxy, s-butoxy, t-butoxy.

“Alkylamino” refers an alkyl group as defined above attached through anamino bridge.

An example of an alkylamino is methylamino, (i.e., —NH—CH₃).

“Aminoalkyl” refers to an amino group attached through an alkyl bridgeas defined above (i.e., NH₂-alkyl-).

“Alkanoyl” refers to an alkyl as defined above attached through acarbonyl bride (i.e., —(C═O)alkyl).

“Alkylsulfonyl” refers to an alkyl as defined above attached through asulfonyl bridge (i.e., —S(═O)₂alkyl) such as mesyl and the like, and“arylsulfonyl” refers to an aryl attached through a sulfonyl bridge(i.e., —S(═O)₂aryl).

“Alkylsulfamoyl” refers to an alkyl as defined above attached through asulfamoyl bridge (i.e., —NHS(═O)₂alkyl), and an “Arylsulfamoyl” refersto an alkyl attached through a sulfamoyl bridge (i.e., —NHS(═O)₂aryl).

“Alkylsulfinyl” refers to an alkyl as defined above attached through asulfinyl bridge (i.e. —S(═O)alkyl).

The terms “halogen” and “halo” refer to fluorine, chlorine, bromine, andiodine.

The term “substituted” refers to a molecule wherein at least onehydrogen atom is replaced with a substituent. When substituted, one ormore of the groups are “substituents.” The molecule may be multiplysubstituted. In the case of an oxo substituent (“═O”), two hydrogenatoms are replaced. Example substituents within this context may includehalogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo, carbocyclyl,carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl, aryl,arylalkyl, heteroaryl, heteroarylalkyl, —NR_(a)R_(b), —NR_(a)C(═O)R_(b),—NR_(a)C(═O)NR_(a)NR_(b), —NR_(a)C(═O)OR_(b), —NR_(a)S₂R_(b),—C(═O)R_(a), —C(═O)OR_(a), —C(═O)NR_(a)R_(b), —OC(═O)NR_(a)R_(b),—OR_(a), —SR_(a), —SOR_(a), —S(═O)₂R_(a), —OS(═O)₂R_(a) and—S(═O)₂R_(a). R_(a) and R_(b) in this context may be the same ordifferent and independently hydrogen, halogen hydroxyl, alkyl, alkoxy,alkyl, amino, alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl,heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl,heteroarylalkyl.

The term “optionally substituted,” as used herein, means thatsubstitution is optional and therefore it is possible for the designatedatom to be unsubstituted.

As used herein, “salts” refer to derivatives of the disclosed compoundswhere the parent compound is modified making acid or base salts thereof.Examples of salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines, alkylamines, ordialkylamines; alkali or organic salts of acidic residues such ascarboxylic acids; and the like. In preferred embodiment the salts areconventional nontoxic pharmaceutically acceptable salts including thequaternary ammonium salts of the parent compound formed, and non-toxicinorganic or organic acids. Preferred salts include those derived frominorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic,phosphoric, nitric and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicylic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, and the like.

“Subject” refers any animal, preferably a human patient, livestock, ordomestic pet.

The term “prodrug” refers to an agent that is converted into abiologically active form in vivo. Prodrugs are often useful because, insome situations, they may be easier to administer than the parentcompound. They may, for instance, be bioavailable by oral administrationwhereas the parent compound is not. The prodrug may also have improvedsolubility in pharmaceutical compositions over the parent drug. Aprodrug may be converted into the parent drug by various mechanisms,including enzymatic processes and metabolic hydrolysis.

As used herein, the term “derivative” refers to a structurally similarcompound that retains sufficient functional attributes of the identifiedanalogue. The derivative may be structurally similar because it islacking one or more atoms, substituted, a salt, in differenthydration/oxidation states, or because one or more atoms within themolecule are switched, such as, but not limited to, replacing an oxygenatom with a sulfur or nitrogen atom or replacing an amino group with ahydroxyl group or vice versa. The derivative may be a prodrug.Derivatives may be prepared by any variety of synthetic methods orappropriate adaptations presented in synthetic or organic chemistry textbooks, such as those provide in March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, Wiley, 6th Edition (2007) MichaelB. Smith or Domino Reactions in Organic Synthesis, Wiley (2006) Lutz F.Tietze hereby incorporated by reference.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity of the disease isreduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. patient) is cured and the disease iseradicated. Rather, embodiments, of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

“Cancer” refers any of various cellular diseases with malignantneoplasms characterized by the proliferation of cells. It is notintended that the diseased cells must actually invade surrounding tissueand metastasize to new body sites. Cancer can involve any tissue of thebody and have many different forms in each body area. Within the contextof certain embodiments, whether “cancer is reduced” may be identified bya variety of diagnostic manners known to one skill in the art including,but not limited to, observation the reduction in size or number of tumormasses or if an increase of apoptosis of cancer cells observed, e.g., ifmore than a 5% increase in apoptosis of cancer cells is observed for asample compound compared to a control without the compound. It may alsobe identified by a change in relevant biomarker or gene expressionprofile, such as PSA for prostate cancer, HER2 for breast cancer, orothers.

Methods of Use

In certain embodiments, the compounds described herein are useful forthe treatment of viral infections where the virus utilizes CXCR4 and/orCCR5 to infect cells.

In one embodiment, the disclosure relates to a method of treating orpreventing HIV infection or reduction of symptoms associated with AIDSis provided including administering a compound disclosed herein to asubject. In certain embodiments, the compound can be provided to asubject before treatment of infection with another compound. In aseparate embodiment, the compound is provided to a patient that has beentreated for HIV infection to reduce the likelihood of recurrence, orreduce mortality associated with AIDS related symptoms. In anotherembodiment, the compound is administered to a subject at high risk ofsuffering from HIV infections.

Subjects, including humans suffering from, or at risk for, HIV infectioncan be treated by administering an effective amount of the activecompound or a pharmaceutically acceptable prodrug or salt thereof in thepresence of a pharmaceutically acceptable carrier or diluent.

The administration can be prophylactically for the prevention of HIVinfection or reduction of symptoms associated with AIDS. The activematerials can be administered by any appropriate route, for example,orally, parenterally, intravenously, intradermally, subcutaneously, ortopically, in liquid or solid form.

In a separate embodiment, a method for the treatment or prevention ofHIV infection or reduction of symptoms associated with AIDS byadministering a compound or derivative of the present disclosure, or apharmaceutically acceptable salt, solvate, prodrug, or ester thereof toa subject in need of treatment is provided. The compounds of thedisclosure, or a pharmaceutically acceptable salt, solvate, prodrug, orester thereof can be administered to a subject in need thereof to reducethe severity of AIDS related disorders. In one embodiment of thedisclosure, the subject is a human.

In a separate embodiment, a method for the treatment of, prevention of,or reduced severity of liver disease associated with viral infectionsincluding administering at least one compound described herein isprovided.

Chronic hepatitis C virus (HCV) and hepatitis B virus (HBC) infection isaccompanied by inflammation and fibrosis eventually leading tocirrhosis. A study testing the expression and function of CXCR4 onliver-infiltrating lymphocytes (LIL) revealed an important role for theCXCL12/CXCR4 pathway in recruitment and retention of immune cells in theliver during chronic HCV and HBV infection (Wald et al., (2004) Eur JImmun; 34(4): 1164-1174). High levels of CXCR4 and TGFβ have beendetected in liver samples obtained from patients infected with HCV.(Mitra et al., (1999) Int J Oncol 14: 917-925). In vitro, TGF-β has beenshown to up-regulate the expression of CXCR4 on T cells and to increasetheir migration. The CD69/TGFβ/CXCR4 pathway may be involved in theretention of recently activated lymphocytes in the liver (Wald et al.,Eur J Immun, 2004; 34(4): 1164-1174).

In another embodiment, the disclosure relates to a method of treatingsymptoms associated with other infections associated with chemokinereceptor activation, for example, liver diseases associated withflavivirus or pestivirus infection, and in particular, HCV or HBV, bycontacting a cell with a compound of the present disclosure, or apharmaceutically acceptable salt, solvate, prodrug, or ester thereof.The cell can be in a subject animal, in particular in a human.

The compounds or derivatives can be used to treat disorders of abnormalcell proliferation generally, examples of which include, but are notlimited to, types of cancers and proliferative disorders listed below.Abnormal cellular proliferation, notably hyperproliferation, can occuras a result of a wide variety of factors, including genetic mutation,infection, exposure to toxins, autoimmune disorders, and benign ormalignant tumor induction.

There are a number of skin disorders associated with cellularhyperproliferation. Psoriasis, for example, is a benign disease of humanskin generally characterized by plaques covered by thickened scales.Chronic eczema is also associated with significant hyperproliferation ofthe epidermis. Other diseases caused by hyperproliferation of skin cellsinclude atopic dermatitis, lichen planus, warts, pemphigus vulgaris,actinic keratosis, basal cell carcinoma and squamous cell carcinoma.Other hyperproliferative cell disorders include blood vesselproliferation disorders, fibrotic disorders, autoimmune disorders,graft-versus-host rejection, tumors and cancers.

Blood vessel proliferative disorders include angiogenic and vasculogenicdisorders. Proliferation of smooth muscle cells in the course ofdevelopment of plaques in vascular tissue cause, for example,restenosis, retinopathies and atherosclerosis. The advanced lesions ofatherosclerosis result from an excessive inflammatory-proliferativeresponse to an insult to the endothelium and smooth muscle of the arterywall (Ross, Nature, 1993, 362:801-809). Both cell migration and cellproliferation play a role in the formation of atherosclerotic lesions.

Fibrotic disorders are often due to the abnormal formation of anextracellular matrix. Examples of fibrotic disorders include hepaticcirrhosis and mesangial proliferative cell disorders. Hepatic cirrhosisis characterized by the increase in extracellular matrix constituentsresulting in the formation of a hepatic scar.

Mesangial disorders are brought about by abnormal proliferation ofmesangial cells. Mesangial hyperproliferative cell disorders includevarious human renal diseases, such as glomerulonephritis, diabeticnephropathy, malignant nephrosclerosis, thrombotic microangiopathysyndromes, transplant rejection, and glomerulopathies.

Another disease with a proliferative component is rheumatoid arthritis.Rheumatoid arthritis is generally considered an autoimmune disease thatis thought to be associated with activity of autoreactive T cells (See,e.g., Harris, (1990) The New England Journal of Medicine,322:1277-1289), and to be caused by auto-antibodies produced againstcollagen and IgE.

Other disorders that can include an abnormal cellular proliferativecomponent include Behcet's syndrome, acute respiratory distress syndrome(ARDS), ischemic heart disease, post-dialysis syndrome, leukemia,acquired immune deficiency syndrome, vasculitis, lipid histiocytosis,septic shock and inflammation in general.

Examples of cancers or proliferative disorders which can be the primarytumor that is treated include but are not limited to neoplasms locatedin the: colon, abdomen, bone, breast, digestive system, liver, pancreas,peritoneum, endocrine glands (adrenal, parathyroid, pituitary,testicles, ovary, thymus, thyroid), eye, head and neck, nervous (centraland peripheral), lymphatic system, pelvis, skin, soft tissue, spleen,thorax, and urogenital tract.

In certain embodiments, the subject is diagnosed with acute childhoodlymphoblastic leukemia; acute lymphoblastic leukemia, acute lymphocyticleukemia, acute myeloid leukemia, adrenocortical carcinoma, adult(primary) hepatocellular cancer, adult (primary) liver cancer, adultacute lymphocytic leukemia, adult acute myeloid leukemia, adultHodgkin's disease, adult Hodgkin's lymphoma, adult lymphocytic leukemia,adult non-Hodgkin's lymphoma, adult primary liver cancer, adult softtissue sarcoma, AIDS-related lymphoma, AIDS-related malignancies, analcancer, astrocytoma, bile duct cancer, bladder cancer, bone cancer,brain stem glioma, brain tumors, breast cancer, cancer of the renalpelvis and ureter, central nervous system (primary) lymphoma, centralnervous system lymphoma, cerebellar astrocytoma, cerebral astrocytoma,cervical cancer, childhood (primary) hepatocellular cancer, childhood(primary) liver cancer, childhood acute lymphoblastic leukemia,childhood acute myeloid leukemia, childhood brain stem glioma, childhoodcerebellar astrocytoma, childhood cerebral astrocytoma, childhoodextracranial germ cell tumors, childhood Hodgkin's disease, childhoodHodgkin's lymphoma, childhood hypothalanic and visual pathway glioma,childhood lymphoblastic leukemia, childhood medulloblastoma, childhoodnon-Hodgkin's lymphoma, childhood pineal and supratentorial primitiveneuroectodermal tumors, childhood primary liver cancer, childhoodrhabdomyosarcoma, childhood soft tissue sarcoma, childhood visualpathway and hypothalamic glioma, chronic lymphocytic leukemia, chronicmyelogenous leukemia, colon cancer, cutaneous T-cell lymphoma, endocrinepancreas islet cell carcinoma, endometrial cancer, ependymoma,epithelial cancer, esophageal cancer, Ewing's sarcoma and relatedtumors, exocrine pancreatic cancer, extracranial germ cell tumor,extragonadal germ cell tumor, extrahepatie bile duct cancer, eye cancer,female Breast cancer, Gaucher's disease, gallbladder cancer, gastriccancer, gastrointestinal carcinoid tumor, gastrointestinal tumors, germcell tumors, gestational trophoblastic tumor, hairy cell leukemia, headand neck cancer, hepatocellular cancer, Hodgkin's disease, Hodgkin'slymphoma, hypergammaglobulinemia, hypopharyngeal cancer, intestinalcancers, intraocular melanoma, islet cell carcinoma, islet cellpancreatic cancer, Kaposi's sarcoma, kidney cancer, laryngeal cancer,lip and oral cavity cancer, liver cancer, lung cancer, lymphoproliferative disorders, macroglobulinemia, male breast cancer,malignant mesothelioma, malignant thymoma, medulloblastomia, melanoma,mesothelioma, metastatie occult primary squamous neck cancer, metastatieprimary squamous neck cancer, metastatie squamous neck cancer, multiplemyeloma, multiple myeloma/plasma cell neoplasm, myelodysplasia syndrome,myelogenous leukemia, myeloid leukemia, myeloproliferative disorders,nasal cavity and paranasal sinus cancer, nasopharyngeal cancer,neuroblastoma, non-Hodgkin's lymphoma during pregnancy, nonmelanoma skincancer, non-small cell lung cancer, occult primary metastatie squamousneck cancer, oropharyngeal cancer, osteo/malignant fibrous sarcoma,osteosarcoma/malignant fibrous histiocytoma, osteosarcoma/malignantfibrous histiocytoma of bone, ovarian epithelial cancer, ovarian germcell tumor, ovarian low malignant potential tumor, pancreatic cancer,paraproteinemias, purpura, parathyroid, cancer, penile cancer,pheochromocytoma, pituitary tumor, plasma cell neoplasm/multiplemyeloma, primary central nervous system lymphoma, primary liver cancer,prostate cancer, rectal cancer, renal cell cancer, renal pelvis andureter cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer,sarcoidosis sarcomas, sezary syndrome, skin cancer, small cell lungcancer, small Intestine cancer, soft tissue sarcoma, squamous neckcancer, stomach cancer, supratentorial primitive neuroectodermal andpineal tumors, T-cell lymphoma, testicular cancer, thymoma, thyroidcancer, transitional cell cancer of the renal pelvis and ureter,transitional renal pelvis and ureter cancer, trophoblastic tumors,ureter and renal pelvis cell cancer, urethial cancer, uterine cancer,uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma,vulvar cancer, Waldenstrom's macroglobulinemia, Wilm's tumor, and anyother hyperproliferative disease located in an organ system listedabove.

In certain embodiments, the compound derivatives disclosed herein can beused to treat or prevent hyperplastic disorders including, but are notlimited to, angiofollicular mediastinal lymph node hyperplasia,angiolymphoid hyperplasia with eosinophilia, atypical melanocytichyperplasia, basal cell hyperplasia, benign giant lymph nodehyperplasia, cementum hyperplasia, congenital adrenal hyperplasia,congenital sebaceous hyperplasia, cystic hyperplasia, cystic hyperplasiaof the breast, denture hyperplasia, ductal hyperplasia, endometrialhyperplasia, fibromuscular hyperplasia, foca epithelial hyperplasia,gingival hyperplasia, inflammatory fibrous hyperplasia, inflammatorypapillary hyperplasia, intravascular papillary endothelial hyperplasia,nodular hyperplasia of prostate, nodular regenerative hyperplasia,pseudoepitheliomatous hyperplasia, senile sebaceous hyperplasia, andverrucous hyperplasia; leukemia (including acute leukemia (e.g., acutelymphocytic leukemia, acute myelocytic leukemia (including myeloblasts,promyelocyte, mylomonocytic, monocytic, and erythroleukemia)) andchronic leukemia (e.g., chronic myelocytic (granulocytic) leukemia andchronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumorsincluding, but not limited to, sarcomas and, carcinomas such asfibrosarcoma, myxosarcoma, fiposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, emangioblastoma, acousticneuroma, oligodendrogliomia, menangioma, melanoma, neuroblastoma, andretinoblastoma.

In a separate embodiment, the disclosure relates to a method for thetreatment of, prevention of, or reduced severity of, age-related maculardegeneration (ARMD) and other pathogenic states involving macularretinal pigment epithelial (RPE) cells by administering at least onecompound or derivative described herein to a subject in need thereof.

CXCR4 plays a role in ocular diseases involving the retina such asage-related macular degeneration (ARMD). The retinal pigment epitheliumhas a major role in the physiological renewal of photoreceptor outersegments in the provision of a transport and storage system fornutrients essential to the photoreceptor layer. The retinal pigmentepithelial (RPE) cells predominantly express CXCR4 receptors. (Crane, etal. (2000) J. Immunol. 165: 4372-4278). The level of CXCR4 mRNAexpression increases upon stimulation with IL-1β or TNFα (Dwinell, etal. (1999) Gastroenterology. 117: 359-367). RPE cells also migrated inresponse to SDF-1α indicating that SDF-1α/CXCR4 interactions maymodulate the effects of chronic inflammation and subretinalneovascularization at the RPE site of the blood-retina barrier.

Age-related macular degeneration is characterized by both primary andsecondary damage of macular RPE cells.

In a separate embodiment, a method for the treatment of, prevention of,or reduced severity of inflammatory disease states, neovascularization,and wound healing including administering at least one compound orderivative described herein to a subject in need thereof. Vascularendothelial cells express a multitude of chemokine receptors, with CXCR4being particularly prominent (Gupta, et al. (1998) J Biol Chem. 273:4282; Volin, et al. (1998) Biochem Biophys Res Commnun. 242: 46).

A RT-PCR based strategy which utilized CXCR4 specific primersdemonstrated that mRNA for the chemokine receptor CXCR4 is expressed notonly in primary cultures and transformed type II alveolar epithelialcells (pneumocytes) but also in a number of epithelial cell linesderived from various other tissues. (Murdoch, et al. (1998) Immunology.98(1): 36-41). Unlike with endothelial cells, CXCR4 is the onlychemokine receptor expressed on epithelial cells. The receptor may havea functional role in epithelial pathology. CXCR4 expressed on theepithelium may facilitate the recruitment of phagocytic cells to sitesof inflammation by direct effects on epithelial cells. CXCR4 may alsohave other functional roles within the immune response or participate inwound healing or neovascularization. CXCR4 may also be involved in thepathophysiology of several acute or chronic inflammatory disease statesassociated with the epithelium.

Certain inflammatory chemokines can be induced during an immune responseto promote cells of the immune system to a site of infection.Inflammatory chemokines function mainly as chemoattractants forleukocytes, recruiting monocytes, neutrophils and other effector cellsfrom the blood to sites of infection or tissue damage. Certaininflammatory chemokines activate cells to initiate an immune response orpromote wound healing. Responses to chemokines include increasing ordecreasing expression of membrane proteins, proliferation, and secretionof effector molecules.

In a particular embodiment, the compounds of the disclosure can beadministered to a host at risk of, or suffering from, an inflammatorycondition. In one embodiment, the compounds are administered for thetreatment or prophylaxis of an inflammatory disorder. In certainembodiments, the inflammatory disorder or condition is mediated bychemokines.

Generally, inflammatory disorders include, but are not limited to,respiratory disorders (including asthma, COPD, chronic bronchitis andcystic fibrosis); cardiovascular related disorders (includingatherosclerosis, post-angioplasty, restenosis, coronary artery diseasesand angina); inflammatory diseases of the joints (including rheumatoidand osteoarthritis); skin disorders (including dermatitis, eczematousdermatitis and psoriasis); post transplantation late and chronic solidorgan rejection; multiple sclerosis; autoimmune conditions (includingsystemic lupus erythematosus, dermatomyositis, polymyositis, Sjogren'ssyndrome, polymyalgia rheumatica, temporal arteritis, Behcet's disease,Guillain Barre, Wegener's granulomatosus, polyarteritis nodosa);inflammatory neuropathies (including inflammatory polyneuropathies);vasculitis (including Churg-Strauss syndrome, Takayasu's arteritis);inflammatory disorders of adipose tissue; and proliferative disorders(including Kaposi's sarcoma and other proliferative disorders of smoothmuscle cells).

In one embodiment, compounds, compositions and methods of treatment ofrespiratory disorders comprising administering a compound as describedherein to a subject in need thereof. Respiratory disorders that may beprevented or treated include a disease or disorder of the respiratorysystem that can affect any part of the respiratory tract. Respiratorydisorders include, but are not limited to, a cold virus, bronchitis,pneumonia, tuberculosis, irritation of the lung tissue, hay fever andother respiratory allergies, asthma, bronchitis, simple and mucopurulentchronic bronchitis, unspecified chronic bronchitis (including chronicbronchitis NOS, chronic tracheitis and chronic tracheobronchitis),emphysema, other chronic obstructive pulmonary disease, asthma, statusasthmaticus and bronchiectasis. Other respiratory disorders includeallergic and non-allergic rhinitis as well as non-malignantproliferative and/or inflammatory disease of the airway passages andlungs.

In one embodiment, the compounds or derivatives of the disclosure areadministered to a patient suffering from a cardiovascular disorderrelated to inflammation. Cardiovascular inflammatory disorders includeatherosclerosis, post-angioplasty, restenosis, coronary artery diseases,angina, and other cardiovascular diseases.

In certain embodiments the disorder is a non-cardiovascular inflammatorydisorder such as rheumatoid and osteoarthritis, dermatitis, psoriasis,cystic fibrosis, post transplantation late and chronic solid organrejection, eczematous dermatitis, Kaposi's sarcoma, or multiplesclerosis. In yet another embodiment, the compounds disclosed herein canbe selected to treat anti-inflammatory conditions that are mediated bymononuclear leucocytes.

In addition, the disclosure is directed to methods of treating animalsubjects, in particular, veterinary and human subjects, to enhance orelevate the number of progenitor cells and/or stem cells. The progenitorand/or stem cells may be harvested and used in cell transplantation. Inone embodiment, bone marrow progenitor and/or stem cells are mobilizedfor myocardial repair. Further, the disclosure is directed to methods oftreating animal subjects, in particular, veterinary and human patients,who are defective in white blood cell (WBQ 8 count, or who would benefitfrom elevation of WBC levels using the compounds disclosed herein.Moreover, the disclosure is directed to methods of effectingregeneration of cardiac tissue in a subject in need of such regenerationusing the disclosed compounds.

The compounds or derivatives of the disclosure may be used for thetreatment of diseases that are associated with immunosuppression such asindividuals undergoing chemotherapy, radiation therapy, enhanced woundhealing and burn treatment, therapy for autoimmune disease or other drugtherapy (e.g., corticosteroid therapy) or combination of conventionaldrugs used in the treatment of autoimmune diseases andgraft/transplantation rejection, which causes immunosuppression;immunosuppression due to congenital deficiency in receptor function orother causes; and infectious diseases, such as parasitic diseases,including but not limited to helminth infections, such as nematodes(round disclosure thus targets a broad spectrum of conditions for whichelevation of progenitor cells and/or stem cells in a subject would bebeneficial or, where harvesting of progenitor cells and/or stem cell forsubsequent stem cell transplantation would be beneficial. In addition,the method of the disclosure targets a broad spectrum of conditionscharacterized by a deficiency in white blood cell count, or which wouldbenefit from elevation of said WBC count.

Formulations Pharmaceutical compositions disclosed herein may be in theform of pharmaceutically acceptable salts, as generally described below.Some preferred, but non-limiting examples of suitable pharmaceuticallyacceptable organic and/or inorganic acids are hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, acetic acid and citricacid, as well as other pharmaceutically acceptable acids known per se(for which reference is made to the references referred to below).

When the compounds of the disclosure contain an acidic group as well asa basic group, the compounds of the disclosure may also form internalsalts, and such compounds are within the scope of the disclosure. When acompound contains a hydrogen-donating heteroatom (e.g. NH), salts arecontemplated to covers isomers formed by transfer of said hydrogen atomto a basic group or atom within the molecule.

Pharmaceutically acceptable salts of the compounds include the acidaddition and base salts thereof. Suitable acid addition salts are formedfrom acids which form non-toxic salts. Examples include the acetate,adipate, aspartate, benzoate, besylate, bicarbonate/carbonate,bisulphate/sulphate, borate, camsylate, citrate, cyclamate, edisylate,esylate, formate, fumarate, gluceptate, gluconate, glucuronate,hexafluorophosphate, hibenzate, hydrochloride/chloride,hydrobromide/bromide, hydroiodide/iodide, isethionate, lactate, malate,maleate, malonate, mesylate, methylsulphate, naphthylate, 2-napsylate,nicotinate, nitrate, orotate, oxalate, palmitate, pamoate,phosphate/hydrogen phosphate/dihydrogen phosphate, pyroglutamate,saccharate, stearate, succinate, tannate, tartrate, tosylate,trifluoroacetate and xinofoate salts. Suitable base salts are formedfrom bases which form non-toxic salts. Examples include the aluminium,arginine, benzathine, calcium, choline, diethylamine, diolamine,glycine, lysine, magnesium, meglumine, olamine, potassium, sodium,tromethamine and zinc salts. Hemisalts of acids and bases may also beformed, for example, hemisulphate and hemicalcium salts. For a review onsuitable salts, see Handbook of Pharmaceutical Salts: Properties,Selection, and Use by Stahl and Wermuth (Wiley-VCH, 2002), incorporatedherein by reference.

The compounds described herein may be administered in the form ofprodrugs. A prodrug can include a covalently bonded carrier whichreleases the active parent drug when administered to a mammaliansubject. Prodrugs can be prepared by modifying functional groups presentin the compounds in such a way that the modifications are cleaved,either in routine manipulation or in vivo, to the parent compounds.Prodrugs include, for example, compounds wherein a hydroxyl group isbonded to any group that, when administered to a mammalian subject,cleaves to form a free hydroxyl group. Examples of prodrugs include, butare not limited to, acetate, formate and benzoate derivatives of alcoholfunctional groups in the compounds. Methods of structuring a compound asprodrugs can be found in the book of Testa and Mayer, Hydrolysis in Drugand Prodrug Metabolism, Wiley (2006). Typical prodrugs form the activemetabolite by transformation of the prodrug by hydrolytic enzymes, thehydrolysis of amide, lactams, peptides, carboxylic acid esters, epoxidesor the cleavage of esters of inorganic acids.

Pharmaceutical compositions for use in the present disclosure typicallycomprise an effective amount of a compound and a suitable pharmaceuticalacceptable carrier. The preparations may be prepared in a manner knownper se, which usually involves mixing the at least one compoundaccording to the disclosure with the one or more pharmaceuticallyacceptable carriers, and, if desired, in combination with otherpharmaceutical active compounds, when necessary under asepticconditions.

Generally, for pharmaceutical use, the compounds may be formulated as apharmaceutical preparation comprising at least one compound and at leastone pharmaceutically acceptable carrier, diluent or excipient and/oradjuvant, and optionally one or more further pharmaceutically activecompounds.

The compounds can be administered by a variety of routes including theoral, ocular, rectal, transdermal, subcutaneous, intravenous,intramuscular or intranasal routes, depending mainly on the specificpreparation used. The compound will generally be administered in an“effective amount”, by which is meant any amount of a compound that,upon suitable administration, is sufficient to achieve the desiredtherapeutic or prophylactic effect in the subject to which it isadministered.

Depending upon the manner of introduction, the compounds describedherein may be formulated in a variety of ways. Formulations containingone or more compounds can be prepared in various pharmaceutical forms,such as granules, tablets, capsules, suppositories, powders, controlledrelease formulations, suspensions, emulsions, creams, gels, ointments,salves, lotions, or aerosols and the like. In another embodiment, theformulation is administered topically. Suitable topical formulationsinclude, but are not limited to, lotions, ointments, creams, and gels.In a preferred embodiment, the topical formulation is a gel. In anotherembodiment, the formulation is administered intranasally.

Formulations containing one or more of the compounds described hereinmay be prepared using a pharmaceutically acceptable carrier composed ofmaterials that are considered safe and effective and may be administeredto an individual without causing undesirable biological side effects orunwanted interactions. The carrier is all components present in thepharmaceutical formulation other than the active ingredient oringredients. As generally used herein “carrier” includes, but is notlimited to, diluents, binders, lubricants, disintegrators, fillers, pHmodifying agents, preservatives, antioxidants, solubility enhancers, andcoating compositions.

Optional pharmaceutically acceptable excipients present in thedrug-containing tablets, beads, granules or particles include, but arenot limited to, diluents, binders, lubricants, disintegrants, colorants,stabilizers, and surfactants. Diluents, also referred to as “fillers,”are typically necessary to increase the bulk of a solid dosage form sothat a practical size is provided for compression of tablets orformation of beads and granules. Suitable diluents include, but are notlimited to, dicalcium phosphate dihydrate, calcium sulfate, lactose,sucrose, mannitol, sorbitol, cellulose, microcrystalline cellulose,kaolin, sodium chloride, dry starch, hydrolyzed starches, pregelatinizedstarch, silicone dioxide, titanium oxide, magnesium aluminum silicateand powdered sugar.

The compositions described herein can be formulation for modified orcontrolled release. Examples of controlled release dosage forms includeextended release dosage forms, delayed release dosage forms, pulsatilerelease dosage forms, and combinations thereof.

The compounds described herein can be administered adjunctively withother active compounds. These compounds include but are not limited toanalgesics, anti-inflammatory drugs, antipyretics, antidepressants,antiepileptics, antihistamines, antimigraine drugs, antimuscarinics,anxioltyics, sedatives, hypnotics, antipsychotics, bronchodilators,anti-asthma drugs, cardiovascular drugs, corticosteroids, dopaminergics,electrolytes, gastro-intestinal drugs, muscle relaxants, nutritionalagents, vitamins, parasympathomimetics, stimulants, anorectics andanti-narcoleptics. “Adjunctive administration”, as used herein, meansthe compounds can be administered in the same dosage form or in separatedosage forms with one or more other active agents.

In certain embodiments, the disclosure relates to compounds disclosedherein, derivatives, prodrugs, esters, or salts and compositionsthereof. Although it is not intended that certain embodiments of thedisclosure be limited by any particular mechanism it is believed thatthese compounds are chemokine CXCR4 and/or CCR5 receptor modulators.

Applicant has identified novel pan-tropic HIV entry inhibitors. Sincethese compounds inhibit the action of host proteins, they should, inprinciple, synergize with any HIV drug that targets a viral protein,thereby expanding the number of new combination therapies that could bedeveloped. This could be particularly important in resource-challengedenvironments where the use of first-line therapies is not economicallyfeasible.

Single agent, pan-tropic chemokine entry inhibitors could:

-   -   (i) prevent M-, MIT- and T-tropic viruses from entering and        establishing infection in immune cells;    -   (ii) exhibit a high barrier to the development of resistance;        and    -   (iii) synergize with HIV drugs that target viral proteins.

The development of a safe and effective pan-tropic HIV entry inhibitorwould greatly expand the patient population that could be treatedrelative to those being treated with combinations including Maraviroc.

The present disclosure will be described in more detail with referenceto the following non-limiting examples. It should be noted that theparticular assays used in the examples section are designed to providean indication of activity.

A replacement of the benzimidazole ring of AMD11070 with atetrahydroquinoline (THQ) ring and hybridization with a piperazine core,a common component of CCR5 antagonists (Briz et al., J AntimicrobChemother, 2006, 57(4)) resulted in the establishment of the PIP seriesof the present application, which generally exhibited EC₅₀ values <50 nMagainst the T-tropic (CXCR4) virus and 2-7 μM against the M-tropic(CCR5) virus. Applicant subsequently hypothesized that CCR5 activitycould be improved upon in this series by including a difluoro side chainsimilar to Maraviroc. Six diastereomers of this new series weresynthesized and, from these, 1 and 2 proved to be the most potent (seeTable 1, below).

TABLE 1 HIV MAGI EC₅₀ Values Compound No. 1 2 HIV_(IIIB): 0.22 μM 0.07μM HIV_(Ba-L): 2.6 μM 2.61 μM CYP2D6: >20 μM >20 μM CYP3A4: >20 μM >20μM HLM:  58% @ 10 min 65% @ 10 min MLM: 100% @ 10 min 95% @ 10 min

Docking studies showed that 2, which binds in a similar pose toMaraviroc (shown in FIG. 1 ), benefits from an important salt bridgebetween its piperazine nitrogen and Glu283, similarly to Maraviroc. Inthe same model, diastereomer 1 was unable to form this salt bridge.Preliminary screening of the ADME properties of 2 was conducted and thedata showed that 2 does not inhibit the cytochrome P450 isoforms, 2D6and 3A4. This is a remarkable improvement over AMD11070, which potentlyinhibited both isoforms at 1 μM (i.e., 100% (2D6) and 35% (3A4)). Inaddition, 2 showed good stability in both human and mouse livermicrosomes. (See FIGS. 4-5 and Table 2).

While addition of Maraviroc's difluoro side chain markedly improved boththe CXCR4 and CCR5 potencies, Applicant believes that the CCR5 activitycan be further improved by structural modifications to lead compound 2that reinforce the active conformation of Maraviroc.

The first modification replaces the core in 2 with a bridged piperazinering to give compound 3 and based on modeling studies, an overlay of 3aligns better with the bioactive conformer of Maraviroc (FIG. 2 ). Theinclusion of an oxetane ring at the bridged piperazine nitrogen, toproduce 4 is also expected to improve potency (FIG. 3 ). The spacearound residue Tyr37 can also be explored by appending heterocycles topiperazines 2 and 3, which will serve as hydrogen bond acceptors toTyr37.

The piperazine core 5 and 6 were both synthesized using previouslydeveloped protocols (Tagat et al., J Med Chem, 2004, 47(10); Armour etal., Chem Biol Drug Des, 2006, 67(4)), and 2 was prepared via atriacetoxyborohydride-mediated reductive amination reaction of 5 and 6,followed by Boc-deprotection with TFA. The same three-step sequence canbe utilized to generate 3. The piperazine moiety of 5 can be exchangedfor a bridged piperazine, which can be prepared in 8-steps from5-methoxy-3,4-dihydro-2H-pyrrole-2-carboxylic acid methyl ester (Åhmanet al., Org Proc Res Dev, 2008, 12(6)).

A reductive amination reaction between commercially availableheterocyclic aldehydes (for example, those depicted as R′) and 2′ or 3′(i.e., protected versions of 2 and 3) should give 7-10 or their bridgedcounterparts 7′-10′. A reductive amination reaction of 3′ and 3-oxetanecarboxaldehyde, followed by deprotection, will produce 4 (shown in FIG.3 ).

Synthesis of Exemplary Compounds

A 500 mL rb flask equipped with a rubber septum and a magnetic stir barwas set under Ar atmosphere and charged with 5 g of acid 11 (13.7 mmol,1 equiv.), 503 mg of DMAP (4.12 mmol, 0.3 equiv.), 3.16 g of EDCI (16.5mmol, 1.2 equiv.), 196 mL of CH₂Cl₂ and 65 mL of dry MeOH. Afterstirring at rt for 12 h, the reaction mixture was quenched by additionof sat. NaHCO₃solution, extracted with CH₂Cl₂ (3×) and dried overNa₂SO₄. The crude product was purified on silica gel column using 0 to50% EA in hexanes as eluent affording 4.86 g (94%) of the product 12 asa clear oil.

A 250 mL rb flask equipped with a rubber septum and magnetic stir barwas charged with 2.28 g of CaCl₂) (20.5 mmol, 1.6 equiv.) followed by4.86 g of 4-benzyl 1-tert-butyl 2-methyl piperazine-1,2,4-tricarboxylate12 (12.8 mmol, 1 equiv.) dissolved in 128 mL of 1:1 mixture of THE andEtOH. After stirring at rt for 20 min, the clear solution was cooled to0° C. and 2.06 g of NaBH₄ (54.6 mmol, 4.25 equiv.) was added and thesuspension was stirred at 0° C. for 30 min. Then the reaction mixturewas allowed to warm to rt and the stirring was continued for 12 h. Thereaction was quenched by addition of 1 M HCl till no bubbling isobserved (pH paper showed neutral solution) and the product is extractedwith diethyl ether (3×), washed with water (2×), brine and dried overNa₂SO₄. The crude product was purified on silica gel column using 10 to30% EA in hexanes as eluent affording 4.20 g (93%) of product 13 as aclear oil.

A 250 mL rb flask equipped with a magnetic stir bar and septum wascharged with 4.10 g of the alcohol 13 (11.7 mmol, 1 equiv.), 8.64 mL ofEt₃N (62.0 mmol, 5.3 equiv.) and 35 mL of CH₂Cl₂. After reaction mixturewas cooled to 0° C., 7.45 g of SO₃*Py (46.8 mmol, 4 equiv.) dissolved in35 mL of DMSO (dissolve in other flask under Ar) was added dropwise andthe reaction mixture was stirred at 0° C. for 3 h. Then the reactionmixture was quenched by addition of sat. NaHCO₃solution, extracted withCH₂Cl₂ (3×), washed with water and brine and dried over Na₂SO₄. Afterthe organics were concentrated, toluene was added and the organicsolvents were removed under vacuum (rotatory evaporator and high vacuum)to remove residual pyridine and Et₃N. The aldehyde 14 was used in thenext step without further purification.

A 250 mL rb flask equipped with a stir bar was charged with 2.60 g of(S)-5,6,7,8-tetrahydroquinolin-8-amine (17.6 mmol, 1.5 equiv.), 4.08 gof the aldehyde 14 (11.7 mmol, 1 equiv.) and 78 mL of DCE. Then 3.97 gof NaBH(OAc)₃ (18.7 mmol, 1.6 equiv.) was added. After stirring at rtfor 12 h, the reaction mixture was quenched by addition of sat. Na₂CO₃solution and the product was extracted with CH₂Cl₂ (3×) and dried overNa₂SO₄. The crude product was purified on silica gel column using EA (toseparate the SM) followed by 20% MeOH in CH₂Cl₂ as eluent affording 5.12g (91%) of the product 15 as yellowish oil.

A 250 mL rb flask equipped with a stir bar was charged with 3.04 g ofthe amine 15 (6.33 mmol, 1 equiv.), 1.78 g of tert-butyl(4-oxobutyl)carbamate (9.49 mmol, 1.5 equiv.) and 31 mL of DCE. Then2.68 g of NaBH(OAc)₃ (12.7 mmol, 2 equiv.) was added. After stirring atrt for 48 h, the reaction was not done. 380 mg of acetic acid (6.33mmol, 1 equiv.) was added and the stirring was continued for 24 h.Reaction was still incomplete. Then 1.78 g of tert-butyl(4-oxobutyl)carbamate (9.49 mmol, 1.5 equiv.) was added and the reactionwas stirred for 12 h. Then 2.68 g of NaBH(OAc)₃ (12.7 mmol, 2 equiv.)was added. After stirring for 12 h, reaction went to completion. Thereaction mixture was quenched by addition of sat. Na₂CO₃ solution andthe product was extracted with CH₂Cl₂ (3×) and dried over Na₂SO₄. Thecrude product was purified on silica gel column using 0-100% EA inhexanes as eluent affording 2.21 g (53%) of the product 16 as a clearoil.

A 100 mL rb flask equipped with a stir bar and rubber septum was chargedwith 1.36 g of the carbamate 16 (2.09 mmol, 1 equiv.), 293 mg of 20 w %of Pd(OH)₂ on carbon (0.417 mmol, 0.2 equiv.) and 25 mL of dry EtOH.Then 526 mg of NH₄OOCH (8.35 mmol, 4 equiv.) was added in one portion.After stirring at rt for 1 h, the reaction mixture was filtered througha celite plug and the celite plug was washed with EtOH. The organicswere concentrated in vacuo (rotatory evaporator) affording 1.06 g (98%)of the product 17 as a yellowish oil.

According to the reference (Organic Process Research & Development 2008,12, 1094 1103; Organic Process Research & Development 2008, 12,1104-1113), the aldehyde 6 was synthesized from4,4-difluorocyclohexanecarboxylic acid and 5.00 g of ethyl(S)-3-amino-3-phenylpropanoate hydrochloride in 4 steps and 44% overallyield affording the product 6 as a white solid.

A 20 mL vial equipped with a stir bar was charged with 0.171 g of thealdehyde 6 (0.580 mmol, 1.2 equiv.), 0.250 g of the amine 17 (0.483mmol, 1 equiv.), 28 μL of CH₃COOH (0.483 mmol, 1 equiv.) and 4.8 mL ofDCE. Then 0.143 g of NaBH(OAc)₃ (0.676 mmol, 1.4 equiv.) was added andthe suspension was stirred at rt for 12 h. The reaction mixture wasquenched by the addition of a saturated NaHCO₃solution, extracted withCH₂Cl₂ (3×) and dried over Na₂SO₄. The crude product is purified onsilica gel column using 0 to 100% EA in hexanes as eluent affording 296mg (77%) of the product 18 as a slightly yellow oil.

A 20 mL vial equipped with a stir bar was charged with 183 mg of theamine 18 (0.230 mmol, 1 equiv.) dissolved in 2.3 mL of CH₂Cl₂. Then0.531 mL of CF₃COOH (6.89 mmol, 30 equiv.) was added. After stirring atrt for 12 h, the reaction mixture was quenched by addition of a 2 N NaOHsolution, extracted with CH₂Cl₂ (3×) and dried over Na₂SO₄. The crudematerial was purified on a silica gel column using 0-45% Solvent 2(Solvent 2=70% CH₂Cl₂, 30% MeOH, 3% NH₄OH) in CH₂Cl₂ as eluent (4 gcolumn) affording 88 mg (64%) of the product 1 as a white foam. ¹H NMR(400 MHz, CDCl₃, ppm) δ: 8.39 (dd, J=4.8, 1.6 Hz, 1H), 8.20 (d, J=7.0Hz, 1H), 7.30-7.23 (m, 3H), 7.20-7.13 (m, 3H), 6.99 (dd, J=7.7, 4.7 Hz,1H), 5.02 (q, J=6.0 Hz, 1H), 3.94 (dd, J=9.6, 5.8 Hz, 1H), 3.01 (d,J=11.6 Hz, 1H), 2.87 (d, J=11.2 Hz, 1H), 2.81-1.55 (m, 33H), 1.46-1.31(m, 4H). ¹³C NMR (100 MHz, CDCl₃, ppm) δ: 173.21, 158.11, 146.81,142.11, 136.44, 134.01, 128.34, 126.82, 125.92, 124.98, 122.59 (t,J=240.7 Hz), 121.46, 61.64, 58.53, 57.16, 55.04, 54.80, 53.23, 52.89,52.25, 45.53, 42.78, 41.81, 32.71 (t, J=24.4 Hz), 31.21, 31.11, 29.20,26.99, 26.09 (d, J=7.8 Hz), 26.05, 25.87 (d, J=9.4 Hz), 21.54. ¹⁹F NMR(376 MHz, CDCl₃, ppm) δ: −94.08 (d, J=236.6 Hz), −101.35 (d, J=236.5Hz). HRMS (ESI+) calcd for C₃₄H₅₁ON₆F₂ ([M+H]⁺): 597.4087. Found:597.4080, error −0.7 ppm. LC-MS (ESI-API, 254 nm) 75-95% MeOH in H₂O(0.1% HCO₂H), 3 min, 1.00 mL/min, C18 (Agilent Zorbax XDB-18, 50 mm×4.6mm, 3.5 μm), m/z=597.2 (M+H), 299.2 (M/2+H), t=0.628 min.

A 250 mL rb flask equipped with a magnetic stir bar was charged with6.89 g of Na₂CO₃ (65.0 mmol, 2.28 equiv.) dissolved in 49 mL of water atambient temperature and 5.70 g of methyl (S)-2-amino-2-phenylacetatehydrochloride (28.6 mmol, 1 equiv.), followed by 25 ml of CH₂Cl₂ and themixture was cooled in ice bath. Then 4.38 g of4,4-difluorocyclohexane-1-carbonyl chloride (24.0 mmol, 0.84 equiv.)dissolved in 13 mL of toluene was added. After stirring at rt for 2 h,the reaction mixture was adjusted to pH 9-10 by the addition of 10 MNaOH. The crude product was extracted with CH₂Cl₂ (3×), washed with 2 MNaOH and water, 2 M HCl solution, water, brine and dried over Na₂SO₄.The organics were concentrated to 60 mL volume and 6 mL of toluene wasadded. The organics were concentrated to 24 mL volume and 24 mL ofheptane was added. The suspension was cooled and the product wasfiltered, washed with heptane affording 6.95 g (78%) of the product 19as a white solid.

A 250 mL rb flask, equipped with magnetic stir bar, reflux condenser andrubber septum, was charged with 3.0 g of the ester 19 (9.63 mmol, 1equiv.) and 0.729 g of NaBH₄ (19.3 mmol, 2 equiv.) and 23 mL of THF.After the mixture was heated to 50° C., 1.97 mL of MeOH (9.64 mmol, 1equiv.) was added dropwise. Then the reaction mixture was heated toreflux and held for 90 min, then cooled to 20° C. and 3.5 mL of acetonewas added. The mixture was stirred for 15 min and 30 mL of CH₂Cl₂ wasadded, followed by 16 mL of 2 M NaOH solution. After stirring for 2 h,the product was extracted with CH₂Cl₂ (3×), washed with brine and driedover Na₂SO₄. The organics were filtered and concentrated affording 2.39g (88%) of the product 20 as a white solid.

Similar to the ref. Tetrahedron: Asymmetry, 2002, 13, 2509-2512, a 250mL rb flask equipped with a magnetic stir bar and rubber septum wascharged with 1.80 g of the alcohol 20 (6.35 mmol, 1 equiv.) and 50 mL ofCH₂Cl₂. Then 5.66 g of Dess-Martin periodinane (13.3 mmol, 2.1 equiv.)was added. After stirring at rt for 1 h, 21 mL of ethyl ether was addedfollowed by 27.7 g of Na₂S₂O₃*5H₂O dissolved in 23 mL of 80% sat.NaHCO₃. Organic layer was separated, and the aqueous phase was extractedwith 48 mL of diethyl ether. Organic solution was washed with 32 mL ofsat. NaHCO₃solution, 32 mL of water (2×) and 32 mL of brine (2×). Afterdrying over MgSO₄, the solvents were partially evaporated (water bathbelow 20° C.). The residue was triturated with hexanes until turbidityand left at rt overnight. Solvents were removed, solid aldehyde productwas filtered, washed with a 4:1 hexane-ether mixture and dried undervacuum affording 1.01 g (56%) of the product 21 as a white solid. About10 mg of the aldehyde 21 was dissolved in 0.5 mL of MeOH and 20 mg ofNaBH₄ was added. After 10 min, 2 M HCl was added till no bubbling wasobserved and the product was extracted with CH₂Cl₂ (2×), washed withwater and dried over Na₂SO₄. The crude product was analyzed on HPLC.HPLC (210, 254 nm) 5% iPrOH in hexanes, 20 min, 0.85 mL/min, (CHIRALCELOD-H, 250 mm×4.6 mm), t=9.556 min (0.17%), t=12.026 min, (98.78%); Thelow yield and high enantiopurity of the aldehyde can be explained due tocrystallization during last steps of the workup in the synthesis of thealdehyde.

A 250 ml rb flask equipped with a magnetic stir bar was charged with5.31 g of Na₂CO₃ (50.1 mmol, 2.28 equiv.) dissolved in 49 mL of water atambient temperature and 4.39 g of methyl (R)-2-amino-2-phenylacetatehydrochloride (22.0 mmol, 1 equiv.), followed by 25 ml of CH₂Cl₂ and themixture was cooled in ice bath. Then 4.38 g of4,4-difluorocyclohexane-1-carbonyl chloride (24.0 mmol, 1.1 equiv.)dissolved in 13 mL of toluene was added. After stirring at rt for 2 h,the reaction mixture was adjusted to pH 9-10 by the addition of 10 MNaOH. The crude product was extracted with CH₂Cl₂ (3×), washed with 2 MNaOH and water, brine and dried over Na₂SO₄. The organics wereconcentrated to 60 mL volume and 6 mL of toluene was added. The organicswere concentrated to 24 mL volume and 24 mL of heptane was added. Thesuspension was cooled and the product was filtered, washed with heptaneaffording 6.15 g (90%) of the product 22 as a white solid.

A 250 mL rb flask, equipped with magnetic stir bar, reflux condenser andrubber septum, was charged with 3.0 g of the ester 22 (9.63 mmol, 1equiv.) and 0.729 g of NaBH₄ (19.3 mmol, 2 equiv.) and 23 mL of THF.After the mixture was heated to 50° C., 1.97 mL of MeOH (9.64 mmol, 1equiv.) was added was added dropwise. Then the reaction mixture washeated to reflux and held for 90 min, then cooled to 20° C. and 3.5 mLof acetone was added. The mixture was stirred for 15 min and 30 mL ofCH₂Cl₂ was added, followed by 16 mL of 2M NaOH solution. After stirringfor 2 h, the product was extracted with CH₂Cl₂ (3×), washed with brineand dried over Na₂SO₄. The organics were filtered and concentratedaffording 2.51 g (92%) of the product 23 as a white solid.

Similar to the ref. Tetrahedron: Asymmetry, 2002, 13, 2509-2512, a 250mL rb flask equipped with a magnetic stir bar and rubber septum wascharged with 1.80 g of the alcohol 23 (6.35 mmol, 1 equiv.) and 50 mL ofCH₂Cl₂. Then 5.66 g of Dess-Martin periodinane (13.3 mmol, 2.1 equiv.)was added. After stirring at rt for 1 h, 21 mL of ethyl ether was addedfollowed by 27.7 g of Na₂S₂O₃*5H₂O dissolved in 23 mL of 80% sat.NaHCO₃. The organic layer was separated, and the aqueous phase wasextracted with 48 mL of diethyl ether. The organic solution was washedwith 32 mL of sat. NaHCO₃solution, 32 mL of water (2×) and 32 mL ofbrine (2×). After drying over MgSO₄, the solvents were partiallyevaporated (water bath below 20° C.). The residue was triturated withhexanes until turbidity and left at rt overnight. Solvents were removed,the product was filtered, washed with a 4:1 hexane-ether mixture anddried under vacuum affording 0.910 g (51%) of the product 24 as a whitesolid. About 10 mg of the aldehyde 24 was dissolved in 0.5 mL of MeOHand 20 mg of NaBH₄ was added. After 10 min, 2 M HCl was added till nobubbling was observed and the product was extracted with CH₂Cl₂ (2×),washed with water and dried over Na₂SO₄. The crude product was analyzedon HPLC. HPLC (210, 254 nm) 5% iPrOH in hexanes, 20 min, 0.85 mL/min,(CHIRALCEL OD-H, 250 mm×4.6 mm), t=9.615 min (94.45%), no other isomer;The low yield and high enantiopurity of the aldehyde can be explaineddue crystallization during last steps of the workup.

A 100 mL rb flask equipped with a stir bar and rubber septum was chargedwith 0.228 g of the aldehyde 21 (0.811 mmol, 1.2 equiv.), 0.350 g of theamine 17 (0.676 mmol, 1 equiv.) and 20 mL of CH₂Cl₂. Immediately 0.860 gof NaBH(OAc)₃ (4.06 mmol, 6 equiv.) was added and the suspension wasstirred at rt for 12 h. During the reaction, an aldol reaction (546 inLC/MS), racemization of the aldehyde, and kinetic resolution proceeded(the amine prefers R aldehyde) affording 1:4 (SIR) of the isomers. Thereaction mixture was quenched by addition of sat. NaHCO₃solution,extracted with CH₂Cl₂ (3×) and dried over Na₂SO₄. The crude product ispurified on silica gel column using 0 to 100% EA in hexanes as eluentaffording 235 mg of unexpected S,S,R-isomer-25 and 208 mg of a mix ofisomers (1:1 ratio). Another separation was performed to obtain 80 mg ofthe S,S,S-isomer-25 as a white foam.

A 2 dram vial equipped with a stir bar was charged with 80 mg of theamine S,S,S-isomer-25 (0.102 mmol, 1 equiv.) dissolved in 1.5 mL ofdioxane. Then 0.256 mL of 12 M HCl (3.07 mmol, 30 equiv.) was added.After stirring at rt for 1 h, the reaction mixture was quenched byaddition of 2 N NaOH solution, extracted with CH₂Cl₂ (3×) and dried overNa₂SO₄. The crude material was purified on silica gel column using 0-45%of Solvent 2 (Solvent 2=70% CH₂Cl₂, 30% MeOH, 5% NH₄OH) in CH₂Cl₂ aseluent affording 48 mg (81%) of the product 26 as a white foam. ¹H NMR(400 MHz, CDCl₃, ppm) δ: 8.43 (dd, J=4.7, 1.8 Hz, 1H), 7.34-7.17 (m,6H), 7.02 (dd, J=7.7, 4.7 Hz, 1H), 6.59 (d, J=4.9 Hz, 1H), 4.84 (dt,J=10.2, 5.2 Hz, 1H), 3.96 (dd, J=9.5, 5.9 Hz, 1H), 2.90 (A of AB,J_(AB)=11.4 Hz, 1H), 2.84-1.61 (m, 32H), 1.47-1.33 (m, 4H). ¹⁹F NMR (376MHz, CDCl₃, ppm) δ: −93.39 (d, J=236.6 Hz), −100.33 (d, J=236.3 Hz).

A 100 mL rb flask equipped with a stir bar and rubber septum was chargedwith 0.228 g of the aldehyde 24 (0.811 mmol, 1.2 equiv.), 0.350 g of theamine 16 (0.676 mmol, 1 equiv.) and 20 mL of CH₂Cl₂. Immediately 0.860 gof NaBH(OAc)₃ (4.06 mmol, 6 equiv) was added and the suspension wasstirred at rt for 12 h. During reaction, an aldol reaction (546 inLC/MS), racemization of the aldehyde, and kinetic resolution proceeded(the amine prefers R aldehyde) affording single R isomer. The reactionmixture was quenched by addition of sat. NaHCO₃ solution, extracted withCH₂Cl₂ (3×) and dried over Na₂SO₄. The crude product is purified onsilica gel column using 0 to 100% EA in hexanes as eluent affording ofS,S,R-isomer-25 226 mg (pure product) and 200 mg of S,S,R-isomer-25 withsome minor impurity.

A 2 dram vial equipped with a stir bar was charged with 125 mg of theamine S,S,R-isomer-25 (0.160 mmol, 1 equiv.) dissolved in 1.5 mL ofdioxane. Then 0.146 mL of 12 M HCl (4.79 mmol, 30 equiv.) was added.After stirring at rt for 1 h, the reaction mixture was quenched byaddition of 2 N NaOH solution, extracted with CH₂Cl₂ (3×) and dried overNa₂SO₄. The crude material was purified on silica gel column using 0-45%of Solvent 2 (Solvent 2=70% CH₂Cl₂, 30% MeOH, 5% NH₄OH) in CH₂Cl₂ aseluent affording 55 mg (59%) of the product 27 as a white foam. ¹H NMR(400 MHz, CDCl₃, ppm) δ: 8.43 (d, J=4.7 Hz, 1H), 7.34-7.18 (m, 6H), 7.02(dd, J=7.7, 4.6 Hz, 1H), 6.64 (d, J=5.1 Hz, 1H), 4.86 (dt, J=10.4, 5.3Hz, 1H), 3.96 (dd, J=9.3, 5.7 Hz, 1H), 2.93 (A of AB, J_(AB)=11.0 Hz,1H), 2.81-1.59 (m, 32H), 1.51-1.33 (m, 4H). ¹⁹F NMR (376 MHz, CDCl₃,ppm) δ: −94.19 (d, J=236.6 Hz), −101.39 (d, J=235.3 Hz).

(2S)-4-Benzyloxycarbonyl-1-tert-butoxycarbonyl-piperazine-2-carboxylicacid (5.0 g, 13.72 mmol) was dissolved in anhydrous THE (70 mL) andcooled to 0° C. with an ice bath. Borane dimethyl sulfide complex (2.6mL, 27.44 mmol) was added dropwise at 0° C., slowly. Then the reactionsolution was left to warm up to room temperature and stirred overnight.The reaction mixture was then cooled to 0° C. with an ice bath andquenched with water, dropwise, and extracted with EtOAc. The aqueousphase was extracted with DCM (2×). Combined organic layers were driedover anhydrous MgSO₄, filtered and evaporated under reduced pressure. Itwas used without further purification.

Compound 28 (4.74 g, 13.53 mmol) was dissolved in anhydrous DCM (45 mL)and triethylamine (7.54 mL, 54.11 mmol) added and the mixture was cooledto 0° C. A solution of pyridine sulfur trioxide (6.46 g, 40.58 mmol) inDMSO (45 mL) was added at 0° C. and stirred for 1 hour. The reaction wasquenched with a saturated NaHCO₃solution and diluted with ether. Theaqueous phase was extracted with ether (3×). Combined organic layerswere extracted with sodium phosphate dibasic (Na₂HPO₄), 1M HCl andbrine; dried over anhydrous Na₂SO₄, filtered and evaporated. The crudeproduct was used for the next step without purification.

(S)-5,6,7,8-Tetrahydroquinolin-8-amine (2.2 g, 14.84 mmol) was dissolvedin DCE (100 mL) and STAB-H (5.72 g, 26.98 mmol) added at roomtemperature. The reaction mixture was stirred for a few minutes then 29(4.7 g, 13.49 mmol) was added and the mixture was stirred at roomtemperature overnight. The reaction mixture was quenched with asaturated NaHCO₃solution. The aqueous phase was extracted with DCM andcombined organic layers were dried over anhydrous MgSO₄, filtered andevaporated. The desired product was purified with column chromatographyusing DCM:MeOH:NH₃ (9:1:0.2) giving 5.74 g (86% yield).

Compound 30 (3.04 g, 6.33 mmol), 1.78 g of tert-butyl(4-oxobutyl)carbamate (1.78 g, 9.49 mmol) and DCE (30 ml). ThenNaBH(OAc)₃ (2.68 g, 12.7 mmol), and acetic acid (0.38 g, 6.33 mmol) wereadded. After stirring at room temperature for 48 h, the reaction mixturewas quenched by addition of a sat. NaHCO₃solution and the product wasextracted with DCM (3×) and dried over anhydrous MgSO₄. The crudeproduct was purified on silica gel column using 0-100% EA in hexanes aseluent affording 2.21 g (53%) as a clear oil.

Compound 31 (1.2 g, 1.84 mmol) was dissolved in ethanol (30.682 mL) andammonium formate (0.46 g, 7.36 mmol) and palladium hydroxide on carbon(0.26 g, 0.3700 mmol) were added at room temperature. The reactionmixture was stirred at room temperature for 1 hour and then filteredover celite and evaporated. The crude product 5 was used for the nextstep without purification.

Compound 5 (0.41 g, 0.79 mmol) and STAB-H (0.34 g, 1.58 mmol) weresuspended in DCM (20 ml) and stirred for a few minutes at roomtemperature and then4,4-difluoro-N-[(1S)-3-oxo-1-phenyl-propyl]cyclohexanecarboxamide 6(0.23 g, 0.7900 mmol) was added and the reaction mixture was stirredovernight. It was quenched with a saturated NaHCO₃solution. The aqueousphase was extracted with DCM and combined organic layers were dried overanhydrous Na₂SO₄, filtered and evaporated. The crude product waspurified with column chromatography starting with DCM and increased thepolarity with DCM:MeOH:NH₃ (9:1:0.2) slowly to 50% as eluent affording0.37 g (59% over two steps) of 32 as clear oil.

Compound 32 (0.19 g, 0.2300 mmol) was dissolved in DCM (5 mL) and TFA(0.36 mL, 4.65 mmol) was added at room temperature. The reaction mixturewas stirred overnight and then basified with 1 N NaOH to pH>12. Theorganic layer was separated and the aqueous layer was extracted with DCM(3×). The combined organic layers were dried over anhydrous MgSO₄,filtered and evaporated. The product was purified with columnchromatography starting with DCM and increased the polarity slowly toDCM:MeOH:NH₃ (8:2:0.6) as eluent affording 85 mg (61% yield) as acolorless foam. ¹H NMR (500 MHz, Chloroform-d) δ 8.41 (dd, J=4.8, 1.8Hz, 1H), 8.26 (d, J=7.0 Hz, 1H), 7.31 (dd, J=7.8, 1.7 Hz, 1H), 7.26 (d,J=7.6 Hz, 1H), 7.19 (d, J=7.4 Hz, 3H), 7.01 (dd, J=7.6, 4.7 Hz, 1H),4.99 (q, J=6.2 Hz, 1H), 4.01 (dd, J=10.2, 6.3 Hz, 1H), 3.05-2.98 (m,2H), 2.88-2.83 (m, 2H), 2.71 (td, J=11.3, 9.3, 3.8 Hz, 6H), 2.51 (dt,J=12.9, 6.3 Hz, 1H), 2.32-2.18 (m, 3H), 2.16-1.98 (m, 6H), 1.99-1.88 (m,4H), 1.85-1.59 (m, 9H), 1.57-1.35 (m, 6H).

Compound 5 (0.43 g, 0.8300 mmol) and STAB-H (0.35 g, 1.66 mmol) weresuspended in DCM and stirred at room temperature for a few minutes. Thenthe aldehyde 21 (0.23 g, 0.8300 mmol) was added. The reaction mixturewas stirred at room temperature overnight. During the reaction, an aldolreaction (546 in LC/MS), racemization of the aldehyde, and kineticresolution proceeded (the amine prefers S aldehyde) affording 1:3 (R/S)of the isomers. The reaction mixture was quenched by addition of sat.NaHCO₃solution, extracted with DCM (3×) and dried over Na₂SO₄. The crudeproduct was purified on silica gel column using 0 to 100% EA in hexanesas eluent affording 185 mg (29% yield) of desired isomer 33 as a whitefoam.

Compound 33 (0.185 g, 0.24 mmol) was dissolved in DCM (5 mL) and TFA(0.23 mL, 4.73 mmol) was added at room temperature. The reaction mixturewas stirred overnight and was then basified with 1 N NaOH to pH>12. Theorganic layer was separated and the aqueous layer was extracted with DCM(3×). Combined organic layers were dried over anhydrous MgSO₄, filteredand evaporated. The crude product was purified with columnchromatography starting with DCM and increasing the polarity slowly toDCM:MeOH:NH₃ (8:2:0.6) as eluent affording 52 mg (38% yield) of compound34 as a white foam. ¹H NMR (500 MHz, Chloroform-d) δ 8.44 (d, J=4.7 Hz,1H), 7.32 (d, J=7.8 Hz, 1H), 7.27 (d, J=4.2 Hz, 4H), 7.19 (hept, J=4.2Hz, 1H), 7.07 (s, 1H), 7.00 (dd, J=7.7, 4.7 Hz, 1H), 4.91 (dt, J=10.8,5.1 Hz, 1H), 4.04 (dd, J=9.9, 6.3 Hz, 3H), 2.97 (dt, J=11.7, 2.6 Hz,1H), 2.89-2.74 (m, 2H), 2.80-2.72 (m, 3H), 2.66 (ddt, J=27.2, 15.5, 7.5Hz, 7H), 2.52 (dd, J=13.4, 5.2 Hz, 1H), 2.42-2.30 (m, 2H), 2.25 (dd,J=13.3, 7.5 Hz, 1H), 2.18-2.01 (m, 4H), 2.01-1.86 (m, 4H), 1.76-1.50 (m,9H).

Compound 5 (0.41 g, 0.7900 mmol) was dissolved in DCM and STAB-H (0.34g, 1.58 mmol) was added to the solution. After stirring for a fewminutes the aldehyde 24 (0.22 g, 0.7900 mmol) was added to the mixture.The reaction was stirred at room temperature overnight. During reaction,an aldol reaction (546 in LC/MS), racemization of the aldehyde, andkinetic resolution proceeded (the amine prefers S aldehyde) affording1:3 (RS) of the isomers. The reaction mixture was quenched by additionof a sat. NaHCO₃solution, extracted with DCM (3×) and dried over Na₂SO₄.The crude product was purified on silica gel column using 0 to 100% EAin hexanes as eluent affording 40 mg (7% yield) of desired isomer 35 asa white foam.

Compound 35 (0.040 g, 0.05 mmol) dissolved in 1.5 mL of dioxane. Then 12M HCl (0.13 ml, 4.79 mmol) was added. After stirring at rt for 1 h, thereaction mixture was quenched by addition of 2 N NaOH solution,extracted with DCM (3×) and dried over Na₂SO₄. The crude product waspurified with column chromatography starting with DCM and increasing thepolarity slowly to DCM:MeOH:NH₃ (8:2:0.6) as eluent affording 3 mg (10%yield) of compound 36 as a colorless foam. ¹H NMR (600 MHz,Chloroform-d) δ 8.32 (d, J=4.6 Hz, 1H), 7.30-7.27 (m, 2H), 7.24 (t,J=7.6 Hz, 2H), 7.15 (t, J=7.4 Hz, 2H), 7.03-6.95 (m, 1H), 6.36 (s, 1H),4.83 (s, 1H), 3.97 (dd, J=10.0, 6.2 Hz, 1H), 3.00 (s, 1H), 2.78-2.66 (m,5H), 2.64-2.58 (m, 1H), 2.58-2.44 (m, 4H), 2.25-2.15 (m, 1H), 2.13-2.02(m, 2H), 2.00-1.95 (m, 1H), 1.94-1.82 (m, 4H), 1.79-1.69 (m, 4H),1.25-1.13 (m, 7H), 0.97 (t, J=7.2 Hz, 3H), 0.85-0.73 (m, 2H).

37: (S)-5,6,7,8-tetrahydroquinolin-8-amine (0.647 g, 4.36 mmol) andtert-butyl2-formyl-4-(4-(trifluoromethyl)benzyl)piperazine-1-carboxylate (1.3 g,3.49 mmol) were dissolved in 1,2-DCE and stirred for a few minutes. ThenSTAB (1.110 g, 5.24 mmol) was added and the reaction was stirredovernight at room temperature. The reaction mixture was quenched withsaturated NaHCO₃solution and extracted with DCM (3×). Combined organiclayers were washed with water then brine; dried over anhydrous MgSO₄,filtered off and evaporated. The crude product was purified with columnchromatography starting with DCM and increasing the polarity withDCM:MeOH:NH₄OH (9:1:0.1) to 50% as eluent affording 1.25 g (71%) of theproduct 37 as a white foam.38U and 38L: Compound 37 (1.25 g, 2.477 mmol) was dissolved in 1,2-DCEand N,N-diboc butyraldehyde (0.890 g, 3.10 mmol) was added and thereaction was stirred for few minutes. Then STAB (0.788 g, 3.72 mmol) wasadded and the reaction mixture was stirred overnight. The reaction wasquenched with saturated NaHCO₃solution and extracted with DCM, combinedorganic layers were washed with water, then brine and dried overanhydrous MgSO₄; then filtered off and evaporated. The diasteromers wereseparated using column chromatography starting with Hexanes andincreasing the polarity with EtOAc.39: Compound 38U (0.340 g, 0.44 mmol) was dissolved in 4 ml DCM and TFA(1.0 ml, 13.15 mmol) added and the mixture stirred overnight at roomtemperature. The reaction mixture was basified with 3.75 N NaOH solution(pH>12-14). Then extracted with DCM many times. Combined organic phaseswere dried over anhydrous MgSO₄; filtered off and evaporated. The crudeproduct was purified with column chromatography starting with DCM andincreased the polarity with DCM:MeOH:NH₄OH (8:2:0.3) as eluent affording0.15 g (72%) of the product 39 as a white foam.40: Compound 38L Reactant 1 (0.5 g, 0.644 mmol) was dissolved in 5 mlDCM and TFA (1.5 ml, 19.33 mmol) added and the reaction stirredovernight at room temperature. The reaction mixture was basified with3.75 N NaOH solution and extracted with DCM many times. Combined organiclayers were dried over anhydrous MgSO₄; filtered off and evaporated. Thecrude product was purified with column chromatography starting with DCMand increasing the polarity with DCM:MeOH:NH₄OH (8:2:0.3) as eluentaffording 0.22 g (72%) of the product 40 as a white foam.

A 50 mL rb flask equipped with an air condenser, rubber septum and amagnetic stir bar was set under Ar atmosphere and charged with 500 mg of1-(tert-butoxycarbonyl)piperazine-2-carboxylic acid (2.17 mmol, 1equiv.), 80.0 mg of DMAP (0.651 mmol, 0.3 equiv.), 500 mg of EDCI (2.61mmol, 1.2 equiv.), 10 mL of CH₂Cl₂ and 3 mL of dry methanol. Thereaction mixture was stirred at 40° C. for 1.5 h and then the reactionwas allowed to cool to rt. After stirring for 20 h, the reaction mixturewas concentrated and the residue was dissolved in CH₂Cl₂. The CH₂Cl₂solution was washed with water (3×). The water solutions were combinedand the product was back-extracted with CH₂Cl₂ (3×). Then the combinedorganics were washed with sat. NaHCO₃solution, brine and dried overNa₂SO₄. The crude product (661 mg) was purified on silica gel columnusing 0 to 5% MeOH in CH₂Cl₂ as eluent affording 419 mg (79%) of theproduct 41 as a colorless oil.

A 25 mL rb flask equipped with a rubber septum and a magnetic stir barwas set under an Ar atmosphere and charged with 840 mg of 1-tert-butyl2-methyl piperazine-1,2-dicarboxylate 42 (3.44 mmol, 1 equiv.) dissolvedin 6.9 mL of CH₂Cl₂. After the reaction mixture was cooled to 0° C.,0.527 mL of TEA (3.78 mmol, 1.1 equiv.) and 0.515 mL of CbzCl (3.61mmol, 1.05 equiv.) were added dropwise and the reaction mixture wasstirred for 30 min. Then the reaction mixture was allowed to warm to rtand stirring was continued for 2 h. The reaction mixture was quenched byaddition of sat. NH₄C1 solution, extracted with CH₂Cl₂ (2×), washed withsat. NaHCO₃solution (2×), brine and dried over Na₂SO₄. The crude productwas purified on silica gel column using 30% EA in hexanes as eluentaffording 986 mg (76%) of the product 42 as a colorless oil.

A 50 mL rb flask equipped with a rubber septum and magnetic stir bar wascharged with 460 mg of CaCl₂) (4.14 mmol, 1.6 equiv.) followed by 980 mgof 4-benzyl 1-tert-butyl 2-methyl piperazine-1,2,4-tricarboxylate (2.59mmol, 1 equiv.) dissolved in 26 mL of 1:1 mixture of THE and EtOH. Afterstirring at rt for 20 min, the clear solution was cooled to 0° C. and416 mg of NaBH₄ (11.0 mmol, 4.25 equiv.) was added and the suspensionwas stirred at 0° C. for 30 min. Then the reaction mixture was allowedto warm to rt and the stirring was continued for 12 h. The reaction wasquenched by addition of 1 M HCl till no bubbling was observed (pH papershowed neutral solution) and the product was extracted with diethylether (3×), washed with water (2×), brine and dried over Na₂SO₄. Thecrude product was purified on silica gel column using 10 to 30% EA inhexanes as eluent affording 891 mg (98%) of product 43 as a clear oil.

A 100 mL rb flask equipped with a rubber septum and magnetic stir barwas charged with 28 mL of CH₂Cl₂ and 0.497 mL of oxalyl chloride (5.68mmol, 2.25 equiv.) and the solution was cooled to −78° C. Then 0.807 mLof DMSO (11.4 mmol, 4.5 equiv.) was added dropwise. After stirring for30 min, 0.885 g of 4-benzyl 1-tert-butyl2-(hydroxymethyl)piperazine-1,4-dicarboxylate 43 (2.53 mmol, 1 equiv.)dissolved in 13 mL of CH₂Cl₂ was added dropwise and the reaction mixturewas stirred for 1 h. Then 1.58 mL of NEt₃ (11.4 mmol, 4.5 equiv.) wasadded dropwise. After stirring at −78° C. for 20 min, the reactionmixture was allowed to warm to 0° C. and the stirring was continued for1 h. The reaction was quenched by addition of sat. NaHCO₃solution andthe product was extracted with CH₂Cl₂ (3×), washed with water, brine anddried over Na₂SO₄. The solution was concentrated and filtered through asilica gel plug using 50% EA in hexanes as eluent. The crude product waspurified on silica gel column using CH₂Cl₂, then 10% EA in CH₂Cl₂ aseluent affording 773 mg (88%) of the product 44 as a slightly yellowoil.

To a 20 mL vial equipped with a rubber septum and stir bar was added 323mg of the aldehyde 44 (0.927 mmol, 1 equiv.) and 206 mg of(S)-5,6,7,8-tetrahydroquinolin-8-amine (1.39 mmol, 1.5 equiv.) dissolvedin 6.2 mL DCE. After stirring at rt for 2.5 h, 331 mg of NaBH(OAc)₃(1.48 mmol, 1.6 equiv.) was added in one portion and the suspension wasstirred at rt for 2 h. Then the reaction mixture was quenched byaddition of 1N K₂CO₃ solution and the product was extracted with CH₂Cl₂(3×), washed with 1N K₂CO₃ solution, brine and dried over Na₂SO₄. Thecrude product was purified on silica gel column using CH₂Cl₂, then 3%MeOH in CH₂Cl₂ as eluent affording 444 mg (100%) of the product 45 as aslightly yellow oil.

A 20 mL vial was charged with 440 mg of the amine 45 (0.916 mmol, 1equiv.) and 526 mg of the aldehyde 47 (1.83 mmol, 2 equiv.), followed byaddition of 126 μL of AcOH (2.11 mmol, 2.3 equiv.) and the mixture wasstirred at rt for 2 h. Then 408 mg of NaBH(OAc)₃ (1.83 mmol, 2 equiv.)was added. After stirring at rt for 1.5 h, the reaction mixture waswashed with 1N K₂CO₃ (3×). The combined aq. layer was extracted withCH₂Cl₂ (3×) and the combined organics were washed with brine and driedover Na₂SO₄. The crude product (1.06 g) was purified on silica gelcolumn using 10-60% EA in hexanes, then 10% MeOH in CH₂Cl₂ as eluentaffording 327 mg (48%) of URf (R,S) isomer of 46U and 333 mg (48%) ofLRf (S,S) isomer of 46L as clear oils.

A 10 mL rb flask equipped with a stir bar and septum was charged with300 mg of ethyl (S)-3-amino-3-phenylpropanoate hydrochloride (1.31 mmol,1 equiv.), 16.0 mg of DMAP (0.131 mmol, 0.1 equiv.) and 419 μL of NEt₃(3.00 mmol, 2.3 equiv.) dissolved in 1.1 mL of CH₂Cl₂ at 0° C. Then 175μL of cyclopentanecarbonyl chloride (1.44 mmol, 1.1 equiv.) was addeddropwise. After stirring at 0° C. for 2 h, the reaction mixture wasquenched by addition of sat. NH₄Cl solution, extracted with Et₂O (3×)and dried over Na₂SO₄. The crude product was purified on silica gelcolumn using 10-20% EA in hexanes as eluent affording 364 mg (96%) ofthe product 48 as a clear oil which crystallizes to white solid.

A 100 mL rb flask equipped with a rubber septum and magnetic stir barwas charged with 141 mg of LiAlH₄ (3.71 mmol, 1 equiv.) and 27 mL THEand the reaction mixture was cooled to 0° C. Then 1.07 g of the ester 48(3.71 mmol, 1 equiv.) dissolved in 10 mL of THF was added. Afterstirring at 0° C. for 15 min, the suspension was quenched by addition ofwater and the product was extracted with diethyl ether (3×), washed withwater, brine and dried over Na₂SO₄. The crude product (1.19 g) waspurified on silica gel column using 50% EA in hexanes, then 5% MeOH inCH₂Cl₂ as eluent affording 809 mg (88%) of product 49 as a white solid.

A 50 mL rb flask equipped with a rubber septum and magnetic stir bar wascharged with 9.8 mL of CH₂Cl₂ and 0.427 mL of oxalyl chloride (4.91mmol, 1.5 equiv.) and the solution was cooled to −78° C. Then 0.696 mLof DMSO (9.81 mmol, 3 equiv.) was added dropwise. After stirring for 20min, 0.908 g of the alcohol 49 (3.27 mmol, 1 equiv.) dissolved in 10.9mL of CH₂Cl₂ was added dropwise and the reaction mixture was stirred for15 min. Then 1.82 mL of NEt₃ (13.1 mmol, 4 equiv.) was added dropwise.After stirring at −78° C. for 5 min, the reaction mixture was allowed towarm to 0° C. The reaction mixture was quenched by addition of sat.NH₄Cl solution and the product was extracted with CH₂Cl₂ (3×), washedwith brine and dried over Na₂SO₄. The crude product was purified onsilica gel column using 30-50% EA in hexanes as eluent affording 429 mg(48%) of the product 50 as a white solid.

A 20 mL vial equipped with a stir bar and septum was charged with 102 mgof the amine 46L (0.136 mmol, 1 equiv.), 24.0 mg of PdCl₂ (0.136 mmol, 1equiv.), 38 μL of triethylamine (0.271 mmol, 2 equiv.) and 1.0 mL ofCH₂Cl₂. Then 50 μL of triethylsilane (0.312 mmol, 2.3 equiv.) was addeddropwise and the solution turned to black suspension. After stirring atrt for 12 h, the reaction mixture was quenched by addition of saturatedNH₄Cl aq. solution, extracted with diethyl ether (3×), washed with sat.NH₄Cl aq. solution, brine and dried over Na₂SO₄. The crude product waspurified on silica gel column using 0-20% MeOH in CH₂Cl₂, then 20% MeOHand 2% NH₄OH in CH₂Cl₂ as eluent affording 74 mg (88%) of the product51L as a clear oil.

A 3 mL vial equipped with a stir bar and septum was charged with 168 mgof the amine 46U (0.223 mmol, 1 equiv.), 40 mg of PdCl₂ (0.223 mmol, 1equiv.) and 62 μL of triethylamine (0.447 mmol, 2 equiv.). Then 1.07 mLof triethylsilane (6.70 mmol, 30 equiv.) was added dropwise and thesolution turned into a black suspension. After stirring at rt for 30min, the reaction mixture was quenched by addition of saturated NH₄Clsolution. After stirring for 30 min, sat. Na₂CO₃ solution was added,extracted with CH₂Cl₂ (3×), washed with brine and dried over Na₂SO₄. Thecrude product was purified on silica gel column using 0-10% MeOH inCH₂Cl₂, then 20% MeOH and 1% NH₄OH in CH₂Cl₂ as eluent affording 137 mg(100%) of the product 51U as a clear oil.

To a 20 mL vial equipped with a septum and stir bar was added 57 mg ofthe aldehyde 50 (0.233 mmol, 1.2 equiv.), and 120 mg of amine 51L (0.194mmol, 1 equiv.) dissolved in 1.9 mL CH₂Cl₂. After adding 16 μL of aceticacid (0.272 mmol. 1.4 equiv.) and stirring at rt for 2 h, 54 mg ofNaBH(OAc)₃ (0.253 mmol, 1.3 equiv.) was added in one portion and thesuspension was stirred at rt for 12 h. Then the reaction mixture wasquenched by addition of 1 N K₂CO₃ solution and the product was extractedwith CH₂Cl₂ (2×), washed with 1 N K₂CO₃ solution, brine and dried overNa₂SO₄. The crude product was purified on silica gel column using30-100% EA in hexanes, then 0.5-1% NH₄OH in EA as eluent affording 124mg (75%) of the product 52L as a clear oil.

To a 20 mL vial equipped with a septum and stir bar was added 48 mg ofthe aldehyde 50 (0.198 mmol, 1.1 equiv.) and 111 mg of amine 51U (0.180mmol, 1 equiv.) dissolved in 1.8 mL CH₂Cl₂. After adding 13 μL of aceticacid (0.234 mmol. 1.3 equiv.) and stirring at rt for 2 h, 48 mg ofNaBH(OAc)₃ (0.225 mmol, 1.25 equiv.) was added in one portion and thesuspension was stirred at rt for 1.5 h. Then the reaction mixture wasquenched by addition of sat. Na₂CO₃ solution and the product wasextracted with CH₂Cl₂ (3×) and dried over Na₂SO₄. The crude product waspurified on silica gel column using 50-100% EA in hexanes, then 1% NH₄OHin EA as eluent affording 107 mg (70%) of the product 52U as a clearoil.

To a 20 mL vial equipped with a septum and stir bar was added 59 mg ofthe amine 52L (0.070 mmol, 1 equiv.) and 1.4 mL CH₂Cl₂. After adding 161μL of CF₃COOH (2.09 mmol, 30 equiv.) and stirring at rt for 12 h, thereaction mixture was quenched by addition of 2 N NaOH solution and theproduct was extracted with CH₂Cl₂ (3×), and dried over Na₂SO₄. The crudeproduct was purified on silica gel column using 0-10% MeOH in CH₂Cl₂,then 30% MeOH in CH₂Cl₂ with 1-2% NH₄OH as eluent affording 28 mg (74%)of the product 53L as a slightly yellow glassy oil. (after scratching apowder is obtained). ¹H NMR (400 MHz, CDCl₃, ppm) δ: 8.44 (dd, J=4.7,1.7 Hz, 1H), 7.95 (d, J=7.2 Hz, 1H), 7.36-7.17 (m, 6H), 7.03 (dd, J=7.7,4.6 Hz, 1H), 5.09 (q, J=6.0 Hz, 1H), 3.98 (dd, J=9.5, 5.7 Hz, 1H), 3.03(dt, J=11.5, 2.8 Hz, 1H), 2.90 (d, J=11.2 Hz, 1H), 2.85-2.45 (m, 14H),2.36 (ddd, J=13.7, 9.2, 4.8 Hz, 1H), 2.25 (dt, J=12.8, 5.1 Hz, 1H),2.14-1.35 (m, 21H).

The same procedure as for the synthesis of 53L. Starting with 63 mg ofthe 52U, 41 mg (100%) of the product 53U as slightly yellow glassy oil(after scratching a powder was obtained). ¹H NMR (400 MHz, CDCl₃, ppm)δ: 8.42 (dd, J=4.8, 1.7 Hz, 1H), 7.89 (d, J=7.4 Hz, 1H), 7.36-7.14 (m,6H), 7.02 (dd, J=7.7, 4.7 Hz, 1H), 5.01 (q, J=6.4 Hz, 1H), 4.01 (dd,J=10.0, 6.2 Hz, 1H), 3.10-2.46 (m, 15H), 2.38-2.26 (m, 2H), 2.22 (dt,J=12.4, 5.6 Hz, 1H), 2.09-1.37 (m, 21H).

To a 20 mL vial equipped with a septum and stir bar was added 190 mg ofthe aldehyde 44 (0.545 mmol, 1 equiv.), 176 mg of amine 55 (0.600 mmol,1.1 equiv.) dissolved in 5.4 mL DCE and 139 mg of NaBH(OAc)₃ (0.654mmol, 1.2 equiv.) was added in one portion and the suspension wasstirred at rt for 1.5 h. Then the reaction mixture was quenched byaddition of sat. NaHCO₃ solution and the product was extracted withCH₂Cl₂ (3×), washed with brine and dried over Na₂SO₄. The crude product(439 mg) was purified on silica gel column using EA as eluent affording289 mg (85%) of the product 54 as a slightly yellow oil.

A 20 mL vial equipped with a stir bar and septum was charged with 227 mgof the amine 54 (0.363 mmol, 1 equiv.), 32 mg of PdCl₂ (0.181 mmol, 0.5equiv.) and 101 μL of NEt₃ (0.725 mmol, 2 equiv.). Then 0.290 μL oftriethylsilane (1.81 mmol, 5 equiv.) was added dropwise and the solutionturned to black suspension. After stirring at rt for 30 min, thereaction mixture was quenched by addition of 1 N K₂CO₃ solution,extracted with CH₂Cl₂ (3×), washed with brine, dried over Na₂SO₄. Thecrude product was purified on silica gel column using 0-10% MeOH inCH₂Cl₂, then 20% MeOH and 1% NH₄OH in CH₂Cl₂ as eluent affording 138 mg(77%) of the product 56 as a clear oil.

To a 20 mL vial equipped with a septum and stir bar was added 76 mg ofthe aldehyde 50 (0.309 mmol, 1.1 equiv.), 132 mg of amine 56 (0.281mmol, 1 equiv.) dissolved in 2.8 mL CH₂Cl₂. After adding 21 μL of aceticacid (0.365 mmol. 1.3 equiv.) and stirring at rt for 1.5 h, 74 mg ofNaBH(OAc)₃ (0.351 mmol, 1.25 equiv.) was added in one portion and thesuspension was stirred at rt for 12 h. Then the reaction mixture wasquenched by addition of sat. NH₄Cl solution and the product wasextracted with CH₂Cl₂ (3×), washed with saturated NaHCO₃ solution, brineand dried over Na₂SO₄. The crude product was purified on silica gelcolumn using 50-100% EA in hexanes, then 2% NH₄OH in EA as eluentaffording 201 mg (100%) of the product 57 as a slightly yellow oil.

To a 20 mL vial equipped with a septum and stir bar was added 105 mg ofthe amine 57 (0.146 mmol, 1 equiv.) and 3.0 mL CH₂Cl₂. After adding 337μL of TFA (4.37 mmol. 30 equiv.) and stirring at rt for 3 h, thereaction mixture was quenched by addition of water, then sat. Na₂CO₃solution and 2 N NaOH solution and the product was extracted with EA(3×), and dried over Na₂SO₄. The crude product was purified on silicagel column using 0-10% MeOH in CH₂Cl₂, then 30% MeOH in CH₂Cl₂ with 3%NH₄OH as eluent affording 40 mg of the product 58 as a slightly yellowglassy oil. (after scratching a powder was obtained). ¹H NMR (400 MHz,CDCl₃, ppm) δ: 8.36 (s, 0.5H), 8.35 (s, 0.5H), 7.80-7.73 (m, 1H),7.44-7.39 (m, 1H), 7.33-7.26 (m, 2H), 7.24-7.18 (m, 3H), 7.11 (dd,J=4.9, 1.3 Hz, 0.5H), 7.09 (dd, J=4.9, 1.4 Hz, 0.5H), 5.08 (q, J=7.4 Hz,1H), 3.80 (A of AB, J_(AB)=13.8 Hz, 1H), 3.70 (B of AB, J_(AB)=12.9 Hz,0.5H), 3.67 (B of AB, J_(AB)=12.8 Hz, 1H), 3.01-2.95 (m, 1H), 2.91-2.42(m, 13H), 2.40 (s, 1.5H), 2.39 (s, 1.5H), 2.40-2.26 (m, 1H), 2.27-2.15(m, 1H), 2.08-1.92 (m, 2H), 1.90-1.27 (m, 15H).

4-Benzyl 1-(tert-butyl)(R)-2-((((S)-5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)piperazine-1,4-dicarboxylate30 (4.9 g, 10.2 mmol) was dissolved in DCE (50 mL) and sodiumtriacetoxyborohydride (5.57 g, 25.49 mmol) added. The reaction wasstirred at room temperature for few minutes then4-(1,3-dioxoisoindolin-2-yl)butanal (2.44 g, 11.22 mmol) was added andthen the reaction was stirred at room temperature overnight. Thereaction mixture was quenched with saturated NaHCO₃solution and aqueousphase was extracted with DCM 3 times. Combined organic layer was driedover anhydrous MgSO₄, filtered off and evaporated. The desired product59 was purified with column chromatography using DCM:MeOH:NH₃ (9:1:0.2)giving 6.96 g (100% yield).

A 500 mL rb flask equipped with a stir bar and rubber septum was chargedwith 6.96 g of the carbamate 59 (10.2 mmol, 1 equiv.), 1.43 g of 20 w %of Pd(OH)₂ on carbon (2.04 mmol, 0.2 equiv.) and 100 mL of dry MeOH(degassed by bubbling Ar for 1 h). Then 2.57 g of NH₄O₂CH (40.8 mmol, 4equiv.) was added in one portion. After stirring at rt for 36 h, thereaction mixture was filtered through a celite plug and the celite plugwas washed with MeOH. The organics were concentrated in vacuo (rotatoryevaporator). Pd catalyst was still present. The crude product wasdissolved in CH₂Cl₂ and washed with 2 N NaOH solution, back-extractedwith CH₂Cl₂ (3×) and dried over Na₂SO₄. The crude product was purifiedby silica gel column (120 g) using 0-30% of solvent 2 (solvent 2=30%MeOH in CH₂Cl₂₊₃% NH₄OH) in CH₂Cl₂ affording 2.70 g (48%) of the product60 as a yellowish foam.

A 250 mL rb flask equipped with a stir bar and septum was charged with2.70 g of the amine 60 (4.93 mmol, 1 equiv.), 1.75 g of(S)-4,4-difluoro-N-(3-oxo-1-phenylpropyl)cyclohexane-1-carboxamide 6(5.92 mmol, 1.2 equiv.), 0.056 mL of CH₃COOH (0.990 mmol, 0.2 equiv.)and 50 mL of CH₂Cl₂. Then 1.78 g of NaBH(OAc)₃ (8.38 mmol, 1.7 equiv.)was added. After stirring at rt for 12 h, the reaction was not done.0.440 g of the aldehyde 6 (1.49 mmol, 0.3 equiv.) and the stirring wascontinued for 12 h. The reaction mixture was quenched by addition ofsat. NaHCO₃solution and sat. Na₂CO₃ sol., and the product was extractedwith CH₂Cl₂ (3×) and dried over Na₂SO₄. The crude product was purifiedon silica gel column (120 g) using 0-100% EA in hexanes as eluentaffording 3.35 g (82%) of the product 61 as a yellowish foam.

A 250 mL rb flask equipped with a stir bar and septum was charged with3.12 g of the amine 61 (3.77 mmol, 1 equiv.) dissolved in 38 mL ofCH₂Cl₂. Then 8.72 mL of CF₃COOH (113 mmol, 30 equiv.) was added. Afterstirring at rt for 5 h, the reaction mixture was quenched by addition ofsat. Na₂CO₃ solution, extracted with CH₂Cl₂ (3×) and dried over Na₂SO₄.The crude material was purified on silica gel column (80 g) using 0 to25% of Solvent 2 (solvent 2=70% CH₂Cl₂, 30% MeOH, 3% NH₄OH) in CH₂Cl₂ aseluent affording 1.73 g of the product 62 as a yellowish foam and 0.841g of the other fraction of the product (broad peaks in NMR) which afterre-purification gave 0.760 g of the product (still broad peaks in NMR).

TheN-[(1S)-3-[(3R)-3-[[4-(1,3-dioxoisoindolin-2-yl)butyl-[(8S)-5,6,7,8-tetrahydroquinolin-8-yl]amino]methyl]piperazin-1-yl]-1-phenyl-propyl]-4,4-difluoro-cyclohexanecarboxamide62 (250 mg, 0.34 mmol) was dissolved in DCM (5 ml) and addedthiazole-5-carbaldehyde (0.04 ml, 0.4100 mmol), acetic acid (0.02 ml,0.3400 mmol) and then sodium triacetoxyborohydride (116.63 mg, 0.55mmol) and stirred overnight at room temperature. The reaction mixturewas quenched with 1N NaOH solution and aqueous phase was extracted withDCM three times. Combined organic layer was dried over anhydrous MgSO₄,filtered off and evaporated. The desired product 63 was purified withcolumn chromatography using 0-20% MeOH in EtOAc.

TheN-[(1S)-3-[(3R)-3-[[4-(1,3-dioxoisoindolin-2-yl)butyl-[(8S)-5,6,7,8-tetrahydroquinolin-8-yl]amino]methyl]piperazin-1-yl]-1-phenyl-propyl]-4,4-difluoro-cyclohexanecarboxamide62 (0.250 g, 0.34 mmol) was dissolved in DCM (5 ml) and addedoxazole-5-carbaldehyde (0.070 g, 0.69 mmol), acetic acid (0.02 ml,0.3400 mmol) and then sodium triacetoxyborohydride (0.150 g, 0.69 mmol)and stirred overnight at room temperature. The reaction mixture wasquenched with 1N NaOH solution and aqueous phase was extracted with DCMthree times. Combined organic layer was dried over anhydrous MgSO₄,filtered off and evaporated. The desired product 64 was purified withcolumn chromatography using 0-20% MeOH in EtOAc to get 0.120 g (43%yield).

TheN-[(1S)-3-[(3R)-3-[[4-(1,3-dioxoisoindolin-2-yl)butyl-[(8S)-5,6,7,8-tetrahydroquinolin-8-yl]amino]methyl]piperazin-1-yl]-1-phenyl-propyl]-4,4-difluoro-cyclohexanecarboxamide62 (250 mg, 0.34 mmol) was dissolved in DCM (5 ml) and addedparaformaldehyde (0.103 g, 3.44 mmol), acetic acid (0.02 ml, 0.34 mmol)and then sodium triacetoxyborohydride (0.150 g, 0.69 mmol) and stirredovernight at room temperature. The reaction mixture was quenched with 1NNaOH solution and aqueous phase was extracted with DCM three times.Combined organic layer was dried over anhydrous MgSO₄, filtered off andevaporated. The desired product 65 was purified with columnchromatography using 0-20% MeOH in EtOAc to get 0.100 g (39% yield).

N-[(1S)-3-[(3R)-3-[[4-(1,3-dioxoisoindolin-2-yl)butyl-[(8S)-5,6,7,8-tetrahydroquinolin-8-yl]amino]methyl]-4-(thiazol-5-ylmethyl)piperazin-1-yl]-1-phenyl-propyl]-4,4-difluoro-cyclohexanecarboxamide63 (0.17 g, 0.2100 mmol) was dissolved in methanol (2 mL) and thenhydrazine hydrate (0.41 mL, 2.06 mmol) was added and the reactionstirred overnight. The reaction mixture was quenched with saturatedNa₂CO₃ solution and extracted with DCM three times. Combined organiclayer was dried over anhydrous MgSO₄, filtered off and evaporated. Theproduct 66 was purified with DCM:MeOH:NH₃ (8:2:0.6) to get 0.075 g(52.4% yield). ¹H NMR (600 MHz, Chloroform-d) δ 8.65 (s, 1H), 8.55 (d,J=4.6 Hz, 1H), 8.43 (d, J=7.5 Hz, 1H), 7.61 (s, 1H), 7.31 (d, J=7.7 Hz,1H), 7.23 (d, J=7.6 Hz, 2H), 7.21-7.18 (m, 2H), 7.11 (t, J=7.2 Hz, 1H),6.97 (dd, J=7.7, 4.7 Hz, 1H), 4.83 (q, J=7.1 Hz, 1H), 4.20 (d, J=14.5Hz, 1H), 4.09-4.00 (m, 1H), 3.53 (d, J=14.5 Hz, 1H), 3.05-2.94 (m, 1H),2.92-2.82 (m, 1H), 2.76-2.63 (m, 6H), 2.55-2.29 (m, 6H), 2.28-2.19 (m,2H), 2.15 (ddd, J=12.4, 9.1, 5.9 Hz, 1H), 2.07-1.88 (m, 8H), 1.84-1.49(m, 11H), 1.44 (s, 1H), 1.18 (d, J=7.9 Hz, 1H).

N-[(1S)-3-[(3R)-3-[[4-(1,3-dioxoisoindolin-2-yl)butyl-[(8S)-5,6,7,8-tetrahydroquinolin-8-yl]amino]methyl]-4-(oxazol-5-ylmethyl)piperazin-1-yl]-1-phenyl-propyl]-4,4-difluoro-cyclohexanecarboxamide64 (0.12 g, 0.15 mmol) was dissolved in methanol (2 mL) and then addedhydrazine hydrate (0.30 mL, 1.5 mmol) and stirred the reactionovernight. The reaction mixture was quenched with saturated Na₂CO₃solution and extracted with DCM three times. Combined organic layer wasdried over anhydrous MgSO₄, filtered off and evaporated. The product 67was purified with DCM:MeOH:NH₃ (8:2:0.6) to get 0.071 g (71.5 yield). ¹HNMR (600 MHz, Chloroform-d) δ 8.49 (d, J=4.6 Hz, 1H), 8.28 (d, J=7.2 Hz,1H), 7.84 (s, 1H), 7.37 (d, J=7.6 Hz, 1H), 7.29 (d, J=7.2 Hz, 3H), 7.25(s, 1H), 7.21 (t, J=7.1 Hz, 1H), 7.05 (dd, J=7.6, 4.7 Hz, 1H), 6.95 (s,1H), 5.01 (q, J=6.6 Hz, 1H), 4.16 (d, J=15.1 Hz, 1H), 4.09 (dd, J=9.3,6.2 Hz, 1H), 3.60 (d, J=15.1 Hz, 1H), 3.39-3.30 (m, 1H), 2.96 (dd,J=13.2, 3.5 Hz, 1H), 2.84-2.75 (m, 3H), 2.75-2.61 (m, 5H), 2.55 (dt,J=13.3, 6.7 Hz, 2H), 2.48-2.37 (m, 2H), 2.37-2.23 (m, 3H), 2.19-2.05 (m,5H), 2.05-1.98 (m, 1H), 1.97-1.83 (m, 5H), 1.80-1.64 (m, 4H), 1.62-1.49(m, 6H).

N-[(1S)-3-[3-[[4-(1,3-dioxoisoindolin-2-yl)butyl-[(8S)-5,6,7,8-tetrahydroquinolin-8-yl]amino]methyl]-4-methyl-piperazin-1-yl]-1-phenyl-propyl]-4,4-difluoro-cyclohexanecarboxamide65 (0.1 g, 0.1300 mmol) was dissolved in methanol (2 mL) and hydrazinehydrate (0.27 mL, 1.35 mmol) was added to the solution and stirred thereaction overnight. The reaction mixture was quenched with saturatedNa₂CO₃ solution and extracted with DCM three times. Combined organiclayer was dried over anhydrous MgSO₄, filtered off and evaporated. Theproduct 68 was purified with DCM:MeOH:NH₃ (8:2:0.6) to get 0.043 g (52%yield). ¹H NMR (600 MHz, Chloroform-d) δ 8.45 (s, 1H), 7.28 (d, J=7.7Hz, 1H), 7.24-7.16 (m, 5H), 7.13 (t, J=6.9 Hz, 1H), 6.96 (dd, J=7.7, 4.7Hz, 1H), 4.92 (q, J=6.5 Hz, 1H), 3.99 (dd, J=9.1, 6.2 Hz, 1H), 3.33-3.22(m, 1H), 2.83-2.54 (m, 10H), 2.52-2.38 (m, 1H), 2.30 (q, J=14.8, 13.7Hz, 1H), 2.25-2.14 (m, 8H), 2.13-2.02 (m, 3H), 2.02-1.96 (m, 3H), 1.91(dd, J=9.6, 4.5 Hz, 1H), 1.86-1.64 (m, 6H), 1.59-1.40 (m, 5H), 1.30-1.15(m, 2H).

N-[(1S)-3-[(3R)-3-[[4-(1,3-dioxoisoindolin-2-yl)butyl-[(8S)-5,6,7,8-tetrahydroquinolin-8-yl]amino]methyl]-4-(thiazol-5-ylmethyl)piperazin-1-yl]-1-phenyl-propyl]-4,4-difluoro-cyclohexanecarboxamide61 (0.750 g, 0.91 mmol) was dissolved in methanol (10 mL) and then addedhydrazine hydrate (1.80 mL, 9.1 mmol) and stirred the reactionovernight. The reaction mixture was quenched with saturated Na₂CO₃solution and extracted with DCM three times. Combined organic layer wasdried over anhydrous MgSO₄, filtered off and evaporated. The product 69was purified with DCM:MeOH:NH₃ (9:1:0.2). It was yielded 0.495 g(78.3%).

Tert-butyl2-[[4-aminobutyl-[(8S)-5,6,7,8-tetrahydroquinolin-8-yl]amino]methyl]-4-[(3R)-3-[(4,4-difluorocyclohexanecarbonyl)amino]-3-phenyl-propyl]piperazine-1-carboxylate69 (0.15 g, 0.2100 mmol) was dissolved in THE (5 mL) at roomtemperature. Then added N,N-diisopropylethylamine (0.08 mL, 0.4600 mmol)and trimethylsilylisocyanate (0.03 g, 0.2500 mmol) and stirred at roomtemperature for overnight. The reaction was poured into water andaqueous phase was extracted with DCM. Combined organic layer was driedover anhydrous MgSO₄, filtered off and evaporated. Compound 70 was usedfor the next step without purification.

The tert-butyl4-[(3R)-3-[(4,4-difluorocyclohexanecarbonyl)amino]-3-phenyl-propyl]-2-[[[(8S)-5,6,7,8-tetrahydroquinolin-8-yl]-(4-ureidobutyl)amino]methyl]piperazine-1-carboxylate70 (0.15 g, 0.2000 mmol) was dissolved in DCM (3 mL) and addedtrifluoroacetic acid (0.31 mL, 4 mmol) and stirred at room temperatureovernight. The reaction was basified with 1N NaOH to pH>10-12 andextracted with DCM three times. combined organic layer was dried overanhydrous MgSO₄, filtered off and evaporated. The product 71 waspurified with column chromatography starting with DCM and increased thepolarity with DCM:MeOH:NH₃ (8:2:0.3) to afford 0.100 g (78%) yield ¹HNMR (600 MHz, Methanol-d4) δ 8.45 (d, J=3.3 Hz, 1H), 7.67 (d, J=8.1 Hz,1H), 7.39-7.29 (m, 5H), 7.29-7.23 (m, 1H), 5.00 (t, J=7.4 Hz, 1H), 4.16(dd, J=10.4, 6.0 Hz, 1H), 3.49-3.38 (m, 1H), 3.33 (p, J=1.6 Hz, 2H),3.30-3.21 (m, 1H), 3.15 (ddt, J=13.2, 6.3, 3.1 Hz, 1H), 3.10-3.05 (m,1H), 3.04 (s, 1H), 3.01-2.83 (m, 3H), 2.81 (d, J=17.1 Hz, 1H), 2.59 (t,J=7.5 Hz, 2H), 2.54-2.34 (m, 5H), 2.26-2.16 (m, 2H), 2.14-2.05 (m, 4H),2.01 (q, J=7.2 Hz, 2H), 1.98-1.86 (m, 2H), 1.85-1.65 (m, 7H), 1.40 (dt,J=7.2, 3.9 Hz, 6H).

The tert-butyl2-[[4-aminobutyl-[(8S)-5,6,7,8-tetrahydroquinolin-8-yl]amino]methyl]-4-[(3R)-3-[(4,4-difluorocyclohexanecarbonyl)amino]-3-phenyl-propyl]piperazine-1-carboxylate69 (0.15 g, 0.2200 mmol) was dissolved in DCE (3 mL) and addedtetrahydro-4H-pyran-4-one (0.03 mL, 0.2800 mmol) and sodiumtriacetoxyborohydride (0.07 g, 0.3200 mmol). The reaction mixture wasstirred at room temperature for overnight. Then reaction mixture wasquenched with Na₂CO₃ solution and extracted with DCM three times.Combined organic layer was dried over anhydrous MgSO₄, filtered off andevaporated. The product 72 was purified with column chromatographystarting with DCM and increased the polarity with DCM:MeOH:NH₃ (9:1:0.2)(0.15 g 83% yield).

The tert-butyl4-[(3R)-3-[(4,4-difluorocyclohexanecarbonyl)amino]-3-phenyl-propyl]-2-[[4-(tetrahydropyran-4-ylamino)butyl-[(8S)-5,6,7,8-tetrahydroquinolin-8-yl]amino]methyl]piperazine-1-carboxylate72 (0.15 g, 0.1900 mmol) was dissolved in DCM (5 mL) and trifluoroaceticacid (0.3 mL, 3.84 mmol) was added and stirred at room temperatureovernight. The reaction mixture was basified with 1N NaOH to pH>10-12and extracted with DCM three times. Combined organic layer was driedover anhydrous MgSO₄, filtered off and evaporated. The product 73 waspurified with column chromatography starting with DCM and increased thepolarity with DCM:MeOH:NH₃ (8:2:0.6) to afford 0.115 g (88%) yield ¹HNMR (600 MHz, Methanol-d4) δ 8.45 (d, J=4.7 Hz, 1H), 7.63 (d, J=7.7 Hz,1H), 7.39-7.31 (m, 4H), 7.31-7.22 (m, 2H), 5.01 (t, J=7.6 Hz, 1H), 4.83(s, 1H), 4.12 (dd, J=10.6, 6.1 Hz, 1H), 4.07-3.97 (m, 2H), 3.44 (q,J=14.4, 12.0 Hz, 3H), 3.22 (d, J=9.4 Hz, 1H), 3.18-3.10 (m, 2H),3.08-2.86 (m, 6H), 2.81 (d, J=16.4 Hz, 1H), 2.68-2.58 (m, 2H), 2.57-2.35(m, 5H), 2.26-2.16 (m, 2H), 2.16-2.04 (m, 3H), 2.04-1.87 (m, 7H),1.88-1.71 (m, 6H), 1.71-1.49 (m, 7H).

A 20 mL vial equipped with a stir bar was charged with 250 mg of theamine 62 (0.340 mmol, 1 equiv.), 35.6 mg of oxetane-3-carbaldehyde(0.410 mmol, 1.2 equiv.), 0.020 mL of CH₃COOH (0.340 mmol, 1 equiv.) and3.4 mL of CH₂Cl₂ and the solution was stirred for 30 min. Then 117 mg ofNaBH(OAc)₃ (0.550 mmol, 1.6 equiv.) was added. After stirring at rt for2 days, the reaction was not done. 18.0 mg of oxetane-3-carbaldehyde(0.205 mmol, 0.6 equiv.) and 58.0 mg of NaBH(OAc)₃ (0.275 mmol, 0.8equiv.) were added and the stirring was continued for 2 h. The reactionmixture was quenched by addition of sat. NaHCO₃solution and sat. Na₂CO₃sol., and the product was extracted with CH₂Cl₂ (3×) and dried overNa₂SO₄. The crude product was purified on silica gel column (24 g) using0-30% MeOH in EA as eluent affording 249 mg (91%) ofN—((S)-3-((R)-3-(((4-(1,3-dioxoisoindolin-2-yl)butyl)((S)-5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)-4-(oxetan-3-ylmethyl)piperazin-1-yl)-1-phenylpropyl)-4,4-difluorocyclohexane-1-carboxamide74 as a white foam.

A 20 mL vial equipped with magnetic stir bar was charged with 137 mg ofthe amine 74 (0.170 mmol, 1 equiv.) and 0.180 mL of the 24% hydrazinesolution in water (1.38 mmol, 8 equiv.) dissolved in 2 mL of MeOH. Afterstirring at rt for 12 h, the reaction mixture was quenched by additionof sat. Na₂CO₃ solution, extracted with CH₂Cl₂ (3×) and dried overNa₂SO₄. The organics were concentrated and the crude product waspurified on silica gel column using 0-60% of solvent 2 (solvent 2=30%MeOH in CH₂Cl₂₊₃% NH₄OH) in CH₂Cl₂ affording 92 mg (80%) ofN—((S)-3-((R)-3-(((4-aminobutyl)((S)-5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)-4-(oxetan-3-ylmethyl)piperazin-1-yl)-1-phenylpropyl)-4,4-difluorocyclohexane-1-carboxamide75 as a white foam. ¹H NMR (400 MHz, CDCl₃, ppm) δ:8.36 (dd, J=4.7, 1.7Hz, 1H), 8.24 (d, J=6.9 Hz, 1H), 7.29 (dd, J=7.8, 1.7 Hz, 1H), 7.27-7.22(m, 2H), 7.19-7.14 (m, 3H), 6.98 (dd, J=7.7, 4.6 Hz, 1H), 5.03 (q, J=6.0Hz, 1H), 4.75 (dd, J=7.8, 6.0 Hz, 1H), 4.71 (dd, J=7.8, 6.0 Hz, 1H),4.36 (t, J=6.2 Hz, 1H), 4.33 (t, J=6.1 Hz, 1H), 3.98 (dd, J=8.7, 5.6 Hz,1H), 3.33 (br s, 1H), 3.27 (dd, J=12.7, 7.9 Hz, 1H), 3.13 (sept., J=7.0Hz, 1H), 2.92 (dd, J=13.2, 3.7 Hz, 1H), 2.79-1.33 (m, 37H). ¹⁹F NMR (376MHz, CDCl₃, ppm) δ: −93.83 (d, J=236.5 Hz), −101.02 (d, J=236.7 Hz).

A 20 mL vial equipped with a stir bar was charged with 250 mg of theamine 62 (0.340 mmol, 1 equiv.), 40.0 mg of 1H-imidazole-4-carbaldehyde(0.410 mmol, 1.2 equiv.), 0.020 mL of acetic acid (0.340 mmol, 1 equiv.)and 3.4 mL of CH₂Cl₂ and the solution was stirred for 1 h. Then 117 mgof NaBH(OAc)₃ (0.550 mmol, 1.6 equiv.) was added. After stirring at rtfor 2 days, the reaction was not complete. 20.0 mg of1H-imidazole-4-carbaldehyde (0.205 mmol, 0.6 equiv.) and 58.0 mg ofNaBH(OAc)₃ (0.275 mmol, 0.8 equiv.) were added and the stirring wascontinued for 12 h. The reaction mixture was quenched by addition ofsat. NaHCO₃solution and sat. Na₂CO₃ sol., and the product was extractedwith CH₂Cl₂ (3×) and dried over Na₂SO₄. The crude product was purifiedon silica gel column (24 g) using 0-30% MeOH in EA as eluent affording177 mg (42%) ofN—((S)-3-((R)-4-((1H-imidazol-5-yl)methyl)-3-(((4-(1,3-dioxoisoindolin-2-yl)butyl)((S)-5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)piperazin-1-yl)-1-phenylpropyl)-4,4-difluorocyclohexane-1-carboxamide76 as a white foam.

A 20 mL vial equipped with magnetic stir bar was charged with 117 mg ofthe amine 76 (0.140 mmol, 1 equiv.) and 0.152 mL of the 24% hydrazinesolution in water (1.16 mmol, 8 equiv.) dissolved in 2 mL of MeOH. Afterstirring at rt for 12 h, the reaction mixture was quenched by additionof sat. Na₂CO₃ solution, extracted with CH₂Cl₂ (3×) and dried overNa₂SO₄. The organics were concentrated and the crude product waspurified on silica gel column using 0-60% of solvent 2 (solvent 2=30%MeOH in CH₂Cl₂₊₃% NH₄OH) in CH₂Cl₂ affording 57 mg (58%) ofN—((S)-3-((R)-4-((1H-imidazol-5-yl)methyl)-3-(((4-aminobutyl)((S)-5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)piperazin-1-yl)-1-phenylpropyl)-4,4-difluorocyclohexane-1-carboxamide77 as a white foam. ¹H NMR (400 MHz, CDCl₃, ppm) δ: 8.39 (dd, J=4.7, 1.6Hz, 1H), 8.02 (s, 1H), 7.63 (s, 1H), 7.39 (dd, J=7.7, 1.6 Hz, 1H),7.30-7.24 (m, 2H), 7.22-7.14 (m, 3H), 7.09 (dd, J=7.7, 4.7 Hz, 1H), 6.91(s, 1H), 4.99 (q, J=6.1 Hz, 1H), 4.17 (dd, J=10.4, 5.7 Hz, 1H), 4.00 (Aof AB, J_(AB)=14.9 Hz, 1H), 3.87 (B of AB, J_(AB)=14.8 Hz, 1H), 3.05(dd, J=14.7, 5.0 Hz, 1H), 2.93-2.57 (m, 8H), 2.50 (t, J=6.8 Hz, 2H),2.54-1.49 (m, 21H), 1.37-1.13 (m, 6H). The imidazole N—H can not beseen. ¹H NMR (600 MHz, CDCl₃, ppm) δ:13.82 (br s, 1H), 8.40 (dd, J=4.8,1.6 Hz, 1H), 7.97 (s, 1H), 7.64 (s, 1H), 7.40 (dd, J=7.7, 1.6 Hz, 1H),7.31-7.25 (m, 2H), 7.22-7.15 (m, 3H), 7.10 (dd, J=7.7, 4.7 Hz, 1H), 6.92(s, 1H), 5.00 (q, J=6.2 Hz, 1H), 4.18 (dd, J=10.8, 5.8 Hz, 1H), 4.02 (d,J=15.1 Hz, 1H), 3.87 (d, J=14.9 Hz, 1H), 3.05 (d, J=14.0 Hz, 1H),2.87-2.60 (m, 8H), 2.51 (d, J=7.1 Hz, 2H), 2.44-1.99 (m, 13H), 1.89-1.53(m, 8H), 1.36-1.17 (m, 6H). ¹⁹F NMR (376 MHz, CDCl₃, ppm) δ: −94.21 (d,J=236.4 Hz), −101.19 (d, J=236.1 Hz).

A 20 mL vial equipped with a stir bar was charged with 238 mg of theamine 62 (0.330 mmol, 1 equiv.), 43.3 mg of1-methyl-1H-imidazole-4-carbaldehyde (0.390 mmol, 1.2 equiv.), 0.019 mLof CH₃COOH (0.330 mmol, 1 equiv.) and 3.4 mL of CH₂Cl₂. Then 111 mg ofNaBH(OAc)₃ (0.520 mmol, 1.6 equiv.) was added. After stirring at rt for12 h, more 43.3 mg of 1-methyl-1H-imidazole-4-carbaldehyde (0.390 mmol,1.2 equiv.) and 56.0 mg of NaBH(OAc)₃ (0.260 mmol, 0.8 equiv.) wereadded and the reaction mixture was stirred for 12 h. Then the reactionmixture was quenched by addition of sat. NaHCO₃solution and sat. Na₂CO₃sol., and the product was extracted with CH₂Cl₂ (3×) and dried overNa₂SO₄. The reaction did not go to completion and was resubmitted toreaction conditions.

A 20 mL vial equipped with a stir bar was charged with the recoveredamine 62 and product mixture (0.330 mmol, 1 equiv.), 43.3 mg of1-methyl-H-imidazole-4-carbaldehyde (0.390 mmol, 1.2 equiv.), 0.019 mLof CH₃COOH (0.330 mmol, 1 equiv.) and 3.4 mL of CH₂Cl₂ and stirred at rtfor 3 h. Then 111 mg of NaBH(OAc)₃ (0.520 mmol, 1.6 equiv.) was added.After stirring at rt for 12 h, the reaction mixture was quenched byaddition of sat. NaHCO₃solution and sat. Na₂CO₃ sol., and the productwas extracted with CH₂Cl₂ (3×) and dried over Na₂SO₄. The crude productwas purified on silica gel column (12 g) using 0-30% MeOH in EA aseluent affording 116 mg (43%) ofN—((S)-3-((R)-3-(((4-(1,3-dioxoisoindolin-2-yl)butyl)((S)-5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)-4-((1-methyl-1H-imidazol-5-yl)methyl)piperazin-1-yl)-1-phenylpropyl)-4,4-difluorocyclohexane-1-carboxamide78 as a yellowish foam.

A 20 mL vial equipped with magnetic stir bar was charged with 106 mg ofthe amine 78 (0.130 mmol, 1 equiv.) and 0.135 mL of the 24% hydrazinesolution in water (1.03 mmol, 8 equiv.) dissolved in 1.3 mL of MeOH.After stirring at rt for 12 h, the reaction mixture was quenched byaddition of sat. Na₂CO₃ solution, extracted with CH₂Cl₂ (3×) and driedover Na₂SO₄.

The organics were concentrated and the crude product was purified onsilica gel column using 0-60% of solvent 2 (solvent 2=30% MeOH inCH₂Cl₂₊₃% NH₄OH) in CH₂Cl₂ affording 73 mg (82%) ofN—((S)-3-((R)-3-(((4-aminobutyl)((S)-5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)-4-((1-methyl-1H-imidazol-5-yl)methyl)piperazin-1-yl)-1-phenylpropyl)-4,4-difluorocyclohexane-1-carboxamide79 as a white foam. ¹H NMR (400 MHz, CDCl₃, ppm) δ: 8.37 (d, J=4.6 Hz,1H), 8.26 (d, J=6.8 Hz, 1H), 7.35 (s, 1H), 7.31 (d, J=7.7 Hz, 1H),7.31-7.21 (m, 2H), 7.23-7.13 (m, 3H), 7.00 (dd, J=7.7, 4.6 Hz, 1H), 6.84(s, 1H), 5.04 (q, J=6.0 Hz, 1H), 4.12 (d, J=13.9 Hz, 1H), 4.01 (dd,J=9.1, 5.7 Hz, 1H), 3.58 (s, 3H), 3.14 (d, J=13.1 Hz, 1H), 3.08 (dd,J=13.4, 3.9 Hz, 1H), 2.87-2.62 (m, 4H), 2.59 (t, J=6.4 Hz, 2H),2.54-1.31 (m, 31H). ¹⁹F NMR (376 MHz, CDCl₃, ppm) δ: −93.89 (d, J=236.3Hz), −100.94 (d, J=235.9 Hz).

A 20 mL vial equipped with a stir bar was charged with 250 mg of theamine 62 (0.340 mmol, 1 equiv.), 55.3 mg of isonicotinaldehyde (0.520mmol, 1.5 equiv.) and 3.4 mL of DCE and the solution was stirred for 1h. Then 117 mg of NaBH(OAc)₃ (0.550 mmol, 1.6 equiv.) was added. Afterstirring at rt for 12 h, the reaction was not done. 55.3 mg ofisonicotinaldehyde (0.520 mmol, 1.5 equiv.) and the stirring wascontinued for 2 h. The reaction mixture was quenched by addition of sat.NaHCO₃and sat. Na₂CO₃ sol., and the product was extracted with CH₂Cl₂(3×) and dried over Na₂SO₄. The crude product was purified on silica gelcolumn (12 g) using 0-30% MeOH in EA as eluent affording 220 mg (78%) ofN—((S)-3-((R)-3-(((4-(1,3-dioxoisoindolin-2-yl)butyl)((S)-5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)-4-(pyridin-4-ylmethyl)piperazin-1-yl)-1-phenylpropyl)-4,4-difluorocyclohexane-1-carboxamide80 as a slightly yellowish foam.

A 20 mL vial equipped with magnetic stir bar was charged with 127 mg ofthe amine 80 (0.160 mmol, 1 equiv.) and 0.203 mL of the 24% hydrazinesolution in water (1.55 mmol, 10 equiv.) dissolved in 1.5 mL of MeOH.After stirring at rt for 2 days, the reaction mixture was quenched byaddition of sat. Na₂CO₃ solution, extracted with CH₂Cl₂ (3×) and driedover Na₂SO₄. The organics were concentrated and the crude product waspurified on silica gel column using 0-60% of solvent 2 (solvent 2=30%MeOH in CH₂Cl₂₊₃% NH₄OH) in CH₂Cl₂ affording 97 mg (91%) ofN—((S)-3-((R)-3-(((4-aminobutyl)((S)-5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)-4-(pyridin-4-ylmethyl)piperazin-1-yl)-1-phenylpropyl)-4,4-difluorocyclohexane-1-carboxamide81 as a white foam. ¹H NMR (400 MHz, CDCl₃, ppm) δ: 8.47-8.43 (m, 2H),8.35 (dd, J=4.8, 1.6 Hz, 1H), 8.29 (d, J=7.0 Hz, 1H), 7.28 (dd, J=7.8,1.7 Hz, 1H), 7.26-7.13 (m, 7H), 6.98 (dd, J=7.7, 4.7 Hz, 1H), 5.02 (q,J=6.2 Hz, 1H), 4.36 (d, J=14.9 Hz, 1H), 4.00 (dd, J=9.1, 5.8 Hz, 1H),3.22 (br s, 1H), 3.10 (d, J=14.9 Hz, 1H), 2.85 (dd, J=13.4, 4.1 Hz, 1H),2.78-1.30 (m, 36H). ¹⁹F NMR (376 MHz, CDCl₃, ppm) δ: −93.84 (d, J=236.4Hz), −100.92 (d, J=236.9 Hz).

Synthesis of Bridged Analogs

A 250 mL rb flask equipped with a stir bar and rubber septum was chargedwith 40.0 g of methyl (S)-5-oxopyrrolidine-2-carboxylate (279 mmol, 1equiv.) and 26.7 mL of dimethylsulfate (279 mmol, 1 equiv.) and thesolution was heated at 56° C. for 18 h. Then the solution was cooled andpoured into 58.4 mL of NEt₃ (419 mmol, 1.5 equiv.) dissolved in 10 mL ofdiethyl ether and the emulsion was stirred for 30 min. Then water (100mL) was added and the product was extracted with diethyl ether (3×),washed with sat. NaHCO₃solution and dried over Na₂SO₄. The organics wereconcentrated and toluene was added. The organics were concentrated again(distills off NEt₃) affording 28.15 g (84 w % in toluene, 23.6 g, 54%)of methyl (S)-5-methoxy-3,4-dihydro-2H-pyrrole-2-carboxylate 82 as abrownish oil.

A 500 mL rb flask equipped with a rubber septum and a magnetic stir barwas charged with 36.0 g of methyl(S)-5-methoxy-3,4-dihydro-2H-pyrrole-2-carboxylate 82 (229 mmol, 1equiv.) and 23.0 mL of methyl nitroacetate (250 mmol, 1.09 equiv.) andthe reaction mixture was stirred at rt for 24 h. The product was notobserved by TLC and the reaction mixture was heated at 60° C. for 40 h.Then solution was cooled and dissolved in CH₂Cl₂ and hexanes. The seedcrystal was added and the formed crystals were filtered and washed withdiethyl ether affording 4.95 g of the product as a yellowish solid. Thefiltrate was washed with water and the product was back extracted withCH₂Cl₂ (2×). The organics were concentrated and the product wascrystallized from CH₂Cl₂ and diethyl ether using seed crystals toinitiate the crystallization affording 4.35 g of the product 83 as ayellow solid. The residue was concentrated and purified on silica gelcolumn (330 g) using 0 to 50% EA in CH₂Cl₂ as eluent affording a yellowoil which was recrystallized from CH₂Cl₂/diethyl ether affording 12.2 gof methyl(S)-5-(2-methoxy-1-nitro-2-oxoethylidene)pyrrolidine-2-carboxylate 83 asa yellowish solid (total 21.5 g, 38%). 1:1 mixture of E/Z isomers:

A 500 mL Parr hydrogenator bottle was charged with 12.2 g of the alkene83 (50.0 mmol, 1 equiv.) and 1.75 g of 20 w % Pd(OH)₂ on carbon (2.50mmol, 0.05 equiv.) dissolved in 30 mL of dry methanol. The mixture wasshaken at 45 psi hydrogen atmosphere for 3 days. The suspension wasfiltered through celite plug and the plug was washed with ethanol. Thesolution was concentrated and the reaction was resubmitted to the samereaction conditions and same workup. The crude product 84 was used inthe next step without further purification.

A 250 mL rb flask equipped with a stir bar was charge with 9.20 g ofmethyl (1R,2R,5S)-4-oxo-3,8-diazabicyclo[3.2.1]octane-2-carboxylate 84(50.0 mmol, 1 equiv.), 11.8 mL of NEt₃ (84.9 mmol, 1.7 equiv.), fewcrystals of DMAP and 100 mL of CH₂Cl₂. Then 16.2 g of CbzOSu (64.9 mmol,1.3 equiv.) was added. After stirring at rt for 48 h, the reactionmixture was quenched by addition of sat. NH₄C1 solution, extracted withCH₂Cl₂ (3×) and dried over Na₂SO₄. The crude product was purified onsilica gel column (120 g) using 0-100% EA in hexanes as eluent affording5.67 g of the product as a yellow oil (still some impurity).

The product 85 was repurified on silica gel column (220 g) using 0 to100% EA in hexanes as eluent affording of 8-benzyl 2-methyl(1R,2R,5S)-4-oxo-3,8-diazabicyclo[3.2.1]octane-2,8-dicarboxylate 85 as aslightly yellow oil. The product was dissolved in toluene and seedcrystals were added. The crystallized product was filtered and washedwith ether affording 4.15 g of the product as yellowish solid. Thefiltrate was concentrated and purified again on silica gel column (80 g)using 0 to 100% EA in CH₂Cl₂ as eluent. The product was crystallizedfrom diethyl ether affording 0.537 g of the product as yellowish solid(total 4.69 g, 29%).

A 500 mL rb flask equipped with a rubber septum and magnetic stir barwas charged with 8.29 g of (C₅H₅)₂ZrHCl (Schwartz reagent) (32.3 mmol,1.2 equiv.) and 200 mL of THF. Then 8.56 g of the amide 85 (26.9 mmol, 1equiv.) dissolved in 100 mL of THF was added dropwise via syringe. Afterstirring the suspension at rt for 30 min, more 2.0 g of the Schwartzreagent (8.07 mmol, 0.3 equiv.). After 10 min stirring, almost clearsolution was obtained. The solution was cooled in ice bath and 9.12 g ofNaBH(OAc)₃ (43.0 mmol, 1.6 equv) was added in one portion. Afterstirring at rt for 2 h, the reaction did not go to completion and more5.70 g of the NaBH(OAc)₃ was added (26.9 mmol, 1 equiv.) and thestirring was continued for 12 h. The reaction mixture was quenched byaddition of sat. NaHCO₃solution, extracted with CH₂Cl₂ (1×), then sat.Na₂CO₃ solution was added to the aqueous phase, extracted with CH₂Cl₂(2×) and the combined organics were dried over Na₂SO₄. The organics wereconcentrated and the crude product was dissolved in CH₂Cl₂ and filteredthrough celite plug. The obtained product (8-benzyl 2-methyl(1R,2R,5S)-3,8-diazabicyclo[3.2.1]octane-2,8-dicarboxylate 86) (yellowoil) was used in the next step without further purification.

A Sample for Characterization was Obtained from Other Experiment:

A 100 mL rb flask equipped with a rubber septum and magnetic stir barwas charged with 650 mg of the Schwartz reagent (2.53 mmol, 1.5 equiv.)and 8 mL of THF. Then 537 mg of the amide (1.69 mmol, 1 equiv.)dissolved in 8 mL of THF was added dropwise via syringe. After stirringthe suspension at rt for 30 min, a clear solution was obtained. Thesolution was cooled in ice bath and 715 mg of NaBH(OAc)₃ (3.37 mmol, 2equv) was added in one portion. After stirring at rt for 12 h, thereaction did not go to completion and more 715 mg of the NaBH(OAc)₃ wasadded (3.37, 2 equiv.) and the stirring was continued for 12 h. Thereaction mixture was quenched by addition of sat. NaHCO₃and sat. Na₂CO₃solutions, extracted with CH₂Cl₂ (3×) and dried over Na₂SO₄. The crudeproduct was purified on silica gel column (12 g) using 0 to 100% EA inhexanes as eluent affording 404 mg (77%) (still some impurity) ofproduct 86 as a yellow oil.

A 250 mL rb flask equipped with a rubber septum and magnetic stir barwas charged with 8.18 g of the amine 86 (26.9 mmol, 1 equiv.), 4.9 mL ofNEt₃ (34.9 mmol, 1.3 equiv., 70 mg of DMAP (0.540 mmol, 0.02 equiv.) and100 mL of CH₂Cl₂. After cooling the reaction mixture in the ice bath,7.04 g of Boc₂O (32.3 mmol, 1.2 equiv.) was added in one portion. Afterstirring the solution at rt for 48 h, more 3.52 g of Boc₂O (16.2 mmol,0.6 equiv.) was added. Then the reaction mixture was quenched byaddition of water, extracted with CH₂Cl₂ (3×) and dried over Na₂SO₄. Theorganics were concentrated and the crude product was purified on silicagel column (120 g) using 0 to 20% EA in CH₂Cl₂ as eluent. The obtainedproduct contained some minor impurities, which were separated on silicagel column (80 g) using 0 to 30% EA in hexanes as eluent affording 4.87g (45%) of 8-benzyl 3-(tert-butyl) 2-methyl(1R,2R,5S)-3,8-diazabicyclo[3.2.1]octane-2,3,8-tricarboxylate 87 as anorange brownish oil.

A 250 mL rb flask equipped with rubber septum and magnetic stir bar wascharged with 3.57 g of the ester 87 (8.83 mmol, 1 equiv.) and 89 mL ofether. The flask was cooled in an ice bath and 13.2 mL of 2M LiBH₄solution in THE (26.5 mmol, 3 equiv.) was added. After stirring at rtfor 20 h, the reaction mixture was quenched by addition of water and 2NHCl solution till the bubbling is done (pH=6), extracted with ethylether (3×) and dried over Na₂SO₄. The crude product was purified onsilica gel column (40 g) using 0 to 50% EA in hexanes as eluentaffording 0.996 g (30%) of 8-benzyl 3-(tert-butyl)(1R,2R,5S)-2-(hydroxymethyl)-3,8-diazabicyclo[3.2.1]octane-3,8-dicarboxylate88 as a clear oil, 0.123 g of the mixture of the product and theby-product and 1.563 g (59%) of more polar by-product (benzyl(6S,9R,9aR)-3-oxohexahydro-1H,3H-6,9-epiminooxazolo[3,4-a]azepine-10-carboxylate).

A 20 mL vial equipped with a magnetic stir bar and septum was chargedwith 980 mg of the alcohol 88 (2.60 mmol, 1 equiv.) 25 mL of CH₂Cl₂.Then 1.66 g of Dess-Martin periodinane (3.90 mmol, 1.5 equiv.) was addedand the reaction mixture was stirred at rt for 1.5 h. Then the reactionmixture was quenched by addition of sat. NaHCO₃and sat. Na₂S₂O₃solutions, extracted with diethyl ether (3×), washed with sat. NaHCO₃andsat. Na₂S₂O₃ solutions and dried over Na₂SO₄. After the organics wereconcentrated and 8-benzyl 3-(tert-butyl)(1R,2R,5S)-2-formyl-3,8-diazabicyclo[3.2.1]octane-3,8-dicarboxylate 89was used in the next step without further purification.

A 250 mL rb flask equipped with a stir bar was charged with 626 mg of(S)-5,6,7,8-tetrahydroquinolin-8-amine (3.38 mmol, 1.3 equiv.), 974 mgof the aldehyde 89 (2.60 mmol, 1 equiv.) and 25 mL of DCE. Then 937 mgof NaBH(OAc)₃ (4.42 mmol, 1.7 equiv.) was added and the suspension wasstirred at rt for 1 h. Then 300 mg more of NaBH(OAc)₃ (1.41 mmol, 0.54equiv.) was added. After stirring at rt for 12 h, the reaction mixturewas quenched by addition of sat NaHCO₃and sat. Na₂CO₃ solutions and theproduct was extracted with CH₂Cl₂ (3×) and dried over Na₂SO₄. The crudeproduct was purified on silica gel column (24 g) using 0 to 15% MeOH inCH₂Cl₂ affording 1.02 g (77%) of 8-benzyl 3-(tert-butyl)(1R,5S)-2-((((S)-5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)-3,8-diazabicyclo[3.2.1]octane-3,8-dicarboxylate90 as a yellowish oil.

A 100 mL rb flask equipped with a stir bar was charge with 1.01 g of theamine 90 (1.98 mmol, 1 equiv.), 0.600 g of4-(1,3-dioxoisoindolin-2-yl)butanal (2.78 mmol, 1.4 equiv.) and 15 mL ofDCE. Then 0.710 g of NaBH(OAc)₃ (3.37 mmol, 1.7 equiv.) was added. Afterstirring at rt for 12 h, the reaction mixture was quenched by additionof sat. Na₂CO₃ solution and the product was extracted with CH₂Cl₂ (3×)and dried over Na₂SO₄. The crude product was purified on silica gelcolumn (24 g) using 0-100% EA in hexanes as eluent affording 1.35 g(96%) of 8-benzyl 3-(tert-butyl)(1R,5S)-2-(((4-(1,3-dioxoisoindolin-2-yl)butyl)((S)-5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)-3,8-diazabicyclo[3.2.1]octane-3,8-dicarboxylate91 as a mixture of the diasteromers as a clear oil. (two diastereomers).

A 100 mL rb flask equipped with a stir bar and rubber septum was chargedwith 1.27 g of the carbamate 91 (1.79 mmol, 1 equiv.), 250 mg of 20 w %of Pd(OH)₂ on carbon (0.360 mmol, 0.2 equiv.) and 18 mL of dry methanol.Then 450 mg of NH₄OOCH (7.18 mmol, 4 equiv.) was added in one portion.After stirring at rt for 5 h, more 450 mg of NH₄OOCH (7.18 mmol, 4equiv.) was added and the reaction mixture was stirred for 12 h. Then450 mg more of NH₄OOCH (7.18 mmol, 4 equiv.) and 250 mg of 20 w % ofPd(OH)₂ on carbon (0.360 mmol, 0.2 equiv.) were added. After stirring atrt for 36 h, the reaction mixture was filtered through a celite plug andthe celite plug was washed with ethanol. The organics were concentratedand the reaction was resubmitted at reaction conditions, rt, 12 h. Theorganics were concentrated in vacuo (rotatory evaporator) and the crudeproduct was dissolved in CH₂Cl₂ and sat. Na₂CO₃ solution was added, theproduct was extracted with CH₂Cl₂ (3×) and dried over Na₂SO₄. The crudeproduct was purified on silica gel column (12 g) using EA, then 30% MeOHin CH₂Cl₂ with 3% of NH₄OH as eluent affording 712 mg (69%) oftert-butyl(1R,5S)-2-(((4-(1,3-dioxoisoindolin-2-yl)butyl)((S)-5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)-3,8-diazabicyclo[3.2.1]octane-3-carboxylate92 as a

A 20 mL vial equipped with a stir bar was charged with 0.472 g of(S)-4,4-difluoro-N-(3-oxo-1-phenylpropyl)cyclohexane-1-carboxamide (1.60mmol, 1.3 equiv.), 0.706 g of the amine 92 (1.23 mmol, 1 equiv.), 70 μLof acetic acid (1.23 mmol, 1 equiv.) and 12.3 mL of CH₂Cl₂. Then 0.443 gof NaBH(OAc)₃ (2.09 mmol, 1.7 equiv.) was added and the suspension wasstirred at rt for 12 h. The reaction mixture was quenched by addition ofsat. NaHCO₃solution, extracted with CH₂Cl₂ (3×) and dried over Na₂SO₄.The crude product was purified on silica gel column (80 g) using EA aseluent affording 640 mg (61%) of the 1st isomer (LRf isomer 93, withsome impurities) as a white foam (re-purifying on 12 g column using 0 to100% EA in hexanes as eluent afforded 492 mg of the product as a whitefoam, still some minor impurities), 80 mg (80%) of the mixture of theisomers and 270 mg (260%) of the 2nd isomer (URf isomer 93) as a whitefoam. On TLC using 15% MeOH in EA, the 3rd isomer moves little bitfaster than 1st isomer.

A 20 mL vial equipped with astir bar was charged with 265 mg of URfisomer 93 (0.310 mmol, 1equiv.) dissolved in 3.0 mL of dioxane. Then0.283 mL of 12 M HCl (9.32 mmol, 30 equiv.) was added. After stirring atrt for 12h, the reaction mixture was quenched by addition of sat. Na₂CO₃solution, extracted with CH₂Cl₂ (3×) and dried over Na₂SO₄. The crudematerial was purified on silica gel column (12 g) using 0 to 2500solvent 2 (solvent 2=7000 CH₂Cl₂, 3000 MeOH, 30% NH₄OH) in CH₂Cl₂ aseluent affording 178 mg (76% o) ofN-((1S)-3-((1R,5S)-2-(((4-(1,3-dioxoisoindolin-2-yl)butyl)((S)-5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-1-phenylpropyl)-4,4-difluorocyclohexane-1-carboxamide(URf isomer 94) as a white foam.

A 20 mL vial equipped with a stir bar was charged with 430 mg of LRfisomer 93 (0.500 mmol, 1 equiv.) dissolved in 5 mL of dioxane. Then0.459 mL of 12 M HCl (15.1 mmol, 30 equiv.) was added. After stirring atrt for 12 h, the reaction mixture was quenched by addition of sat.Na₂CO₃ solution, extracted with CH₂Cl₂ (3×) and dried over Na₂SO₄. Thecrude material was purified on silica gel column (12 g) using 0 to 30%solvent 2 (solvent 2=70% CH₂Cl₂, 30% MeOH, 3% NH₄OH) in CH₂Cl₂ as eluentaffording 171 mg (some impurities) and 182 mg (minor impurities) of theproduct. A second purification was performed with combined fractions onsilica gel column (24 g) using 0 to 30% solvent 2 (solvent 2=70% CH₂Cl₂,30% MeOH, 3% NH₄OH) in CH₂Cl₂ as eluent affording 236 mg (62% yield,contains 1% of unknown isomer) ofN-((1S)-3-((1R,5S)-2-(((4-(1,3-dioxoisoindolin-2-yl)butyl)((S)-5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-1-phenylpropyl)-4,4-difluorocyclohexane-1-carboxamide(LRf isomer 94) as a white foam.

A 20 mL vial equipped with magnetic stir bar was charged with 90.0 mg ofURf isomer 94 (0.120 mmol, 1 equiv.) and 0.651 mL of the 24% hydrazinesolution in water (1.20 mmol, 10 equiv.) dissolved in 1.2 mL ofmethanol. After stirring at rt for 12 h, the reaction mixture wasquenched by addition of 2M KOH sol., extracted with CH₂Cl₂ (3×) anddried over Na₂SO₄. The organics were concentrated and the crude productwas purified on silica gel column (12 g) using 0-60% of solvent 2(solvent 2=30% MeOH in CH₂Cl₂+3% NH₄OH) in CH₂Cl₂ affording 63 mg (85%)ofN-((1S)-3-((1R,5S)-2-(((4-aminobutyl)((S)-5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-1-phenylpropyl)-4,4-difluorocyclohexane-1-carboxamide(URf isomer 95) as a white foam. For URf isomer 95: ¹H NMR (400 MHz,CDCl₃, ppm) δ: 8.85 (d, J=6.8 Hz, 1H), 8.42 (dd, J=4.7, 1.8 Hz, 1H),7.34-7.14 (m, 6H), 7.01 (dd, J=7.7, 4.7 Hz, 1H), 5.02 (q, J=6.7 Hz, 1H),4.02 (dd, J=9.6, 6.2 Hz, 1H), 3.14 (d, J=6.3 Hz, 1H), 3.00 (d, J=6.2 Hz,1H), 2.96-2.87 (m, 2H), 2.80-1.21 (m, 37H).

A 20 mL vial equipped with magnetic stir bar was charged with 90.0 mg ofLRf isomer 94 (0.120 mmol, 1 equiv.) and 0.651 mL of the 24% hydrazinesolution in water (1.20 mmol, 10 equiv.) dissolved in 1.2 mL ofmethanol. After stirring at rt for 12 h, the reaction mixture wasquenched by addition of 2M KOH solution, extracted with CH₂Cl₂ (3×) anddried over Na₂SO₄. The organics were concentrated and the crude productwas purified on silica gel column (12 g) using 0-100% of solvent 2(solvent 2=30% MeOH in CH₂Cl₂₊₃% NH₄OH) in CH₂Cl₂ as eluent. The productis too polar and the column is too large. All fractions were combinedand the purification was repeated on 4 g column affording 45 mg (60%) ofN-((1S)-3-((1R,5S)-2-(((4-aminobutyl)((S)-5,6,7,8-tetrahydroquinolin-8-yl)amino)methyl)-3,8-diazabicyclo[3.2.1]octan-8-yl)-1-phenylpropyl)-4,4-difluorocyclohexane-1-carboxamide(LRf isomer 95) as a yellowish foam.

For LR_(f) isomer 95: ¹H NMR (400 MHz, CDCl₃, ppm) δ:8.40 (d, J=4.7 Hz,1H), 7.41 (d, J=7.7 Hz, 1H), 7.31-7.06 (m, 6H), 6.54 (br s, 1H), 5.01(q, J=7.6 Hz, 1H), 3.92-3.83 (m, 1H), 3.10-2.57 (m, 11H), 2.45 (d,J=13.7 Hz, 1H), 2.38-1.42 (m, 29H).

Synthesis of Opened Top-Piece Analogs

A 100 mL rb flask equipped with a stir bar was charge with 1.31 g of thealdehyde 29 (3.76 mmol, 1 equiv.), 1.34 g of the amine 96 (see synthesisin ACS Med. Chem. Lett., 2018, 9, 17-22) (4.32 mmol, 1.15 equiv.), 1.20g of NaBH(OAc)₃ (5.64 mmol, 1.5 equiv.) and 15 mL of CH₂Cl₂ and thesuspension was stirred at rt for 12 h. Then the reaction mixture wasquenched by addition of sat. NaHCO₃sol., and the product was extractedwith CH₂Cl₂ (3×) and dried over Na₂SO₄. The crude product was purifiedon silica gel column (80 g) using 0-70% EA in hexanes as eluentaffording 1.85 g (77%) of 4-benzyl 1-(tert-butyl)(S)-2-(((4-(1,3-dioxoisoindolin-2-yl)butyl)(pyridin-2-ylmethyl)amino)methyl)piperazine-1,4-dicarboxylate97 as a yellow oil. Later, when the bottom chain is installed,racemization got identified.

A 100 mL rb flask equipped with a stir bar and rubber septum was chargedwith 1.80 g of the carbamate 97 (2.80 mmol, 1 equiv.), 390 mg of 20 w %of Pd(OH)₂ on carbon (0.560 mmol, 0.2 equiv.) and 20 mL of dry ethanol.Then 710 mg of NH₄OOCH (11.2 mmol, 4 equiv.) was added in one portionand heated with heat-gun till bubbling to initiate the catalyst. Afterstirring at rt for 5 h, more 710 mg of NH₄OOCH (11.2 mmol, 4 equiv.) wasadded and the reaction mixture was stirred for 12 h. Then more 710 mg ofNH₄OOCH (11.2 mmol, 4 equiv.) was added. After stirring at rt for 24 h,the reaction mixture was filtered through celite plug and the celiteplug was washed with ethanol. The organics were concentrated in vacuo(rotatory evaporator) and the crude product was dissolved in CH₂Cl₂ andsat. Na₂CO₃ solution was added, the product was extracted with CH₂Cl₂(3×) and dried over Na₂SO₄. The crude product was purified on silica gelcolumn (30 g) using 0 to 30% Solv2 (Solv2=30% MeOH in CH₂Cl₂ with 3%NH₄OH) in CH₂Cl₂ as eluent affording 841 mg (59%) of tert-butyl(R)-2-(((4-(1,3-dioxoisoindolin-2-yl)butyl)(pyridin-2-ylmethyl)amino)methyl)piperazine-1-carboxylate98 as a yellow oil. TLC conditions: 20% MeOH in EA.

A 100 mL rb flask equipped with a stir bar was charged with 0.610 g of(S)-4,4-difluoro-N-(3-oxo-1-phenylpropyl)cyclohexane-1-carboxamide 6(2.07 mmol, 1.3 equiv.), 0.810 g of the amine 98 (1.60 mmol, 1 equiv.),18 μL of acetic acid (0.320 mmol, 0.2 equiv.) and 10 mL of DCE. Then0.570 g of NaBH(OAc)₃ (2.71 mmol, 1.7 equiv.) was added and thesuspension was stirred at rt for 5 h. The reaction mixture was quenchedby addition of sat. Na₂CO₃ solution, extracted with CH₂Cl₂ (3×) anddried over Na₂SO₄. The crude product is purified on silica gel column(40 g) using 0 to 100% EA in hexanes as eluent affording 0.924 g (74%)of tert-butyl(R)-4-((S)-3-(4,4-difluorocyclohexane-1-carboxamido)-3-phenylpropyl)-2-(((4-(1,3-dioxoisoindolin-2-yl)butyl)(pyridin-2-ylmethyl)amino)methyl)piperazine-1-carboxylate99 as a white foam. ˜3:1 d.r. was noticed by ¹³C NMR.

A 20 mL vial equipped with a stir bar was charged with 530 mg of theamine 99 (0.664 mmol, 1 equiv.) dissolved in 6.6 mL of CH₂Cl₂. Then 1.56mL of CF₃COOH (20.1 mmol, 30 equiv.) was added. After stirring at rt for5 h, the reaction mixture was cooled in an ice bath and quenched byaddition of 2 N NaOH solution till pH=11, extracted with CH₂Cl₂ (3×) anddried over Na₂SO₄. The crude material was purified on silica gel columnusing 0-50% Solvent 2 (solvent 2=70% CH₂Cl₂, 30% MeOH, 3% NH₄OH) inCH₂Cl₂ as eluent affording 413 mg (89%) of N-((1S)-3-(3-(((4-(1,3-dioxoisoindolin-2-yl)butyl)(pyridin-2-ylmethyl)amino)methyl)piperazin-1-yl)-1-phenylpropyl)-4,4-difluorocyclohexane-1-carboxamide100 as a yellowish foam.

A 20 mL vial equipped with magnetic stir bar was charged with 65.8 mg oftriphosgene (0.229 mmol, 0.410 equiv.) and 5.4 mL of CH₂Cl₂. Then 137 mgof (R)-1-(9H-fluoren-9-yl)ethan-1-ol (0.650 mmol, 1.2 equiv.) was added,followed by 52.0 μL of pyridine (0.650 mmol, 1.2 equiv.) at 0° C. Afterstirring at rt for 2 h, the reaction mixture was washed with water (3×)and dried over Na₂SO₄. The organics were concentrated and dissolved in4.0 mL dry CH₂Cl₂. Then 373 mg of the amine 100 (0.540 mmol, 1 equiv.)dissolved in 1.4 mL of CH₂Cl₂ was added at 0° C., followed by 120 μL ofNEt(iPr)₂ (0.710 mmol, 1.3 equiv.) and the stirring was continued at rtfor 12h. The reaction mixture was diluted with CH₂Cl₂, washed withwater, brine and dried over Na₂SO₄. The crude material was purified onsilica gel column (10 g) using 0-100% EA in hexanes as eluent affording337 mg (10:1 d.r.) and 174 mg (1:1 d.r.) of the product. The firstfraction was repurified affording 189 mg (38%) of(R)-1-(9H-fluoren-9-yl)ethyl(R)-4-((S)-3-(4,4-difluorocyclohexane-1-carboxamido)-3-phenylpropyl)-2-(((4-(1,3-dioxoisoindolin-2-yl)butyl)(pyridin-2-ylmethyl)amino)methyl)piperazine-1-carboxylate101 as a white foam and 79 mg of a mixed fraction.

A 20 mL vial equipped with a magnetic stir bar was charged with 250 mgof the amine 101 (0.260 mmol, 1 equiv.) and 2.6 mL of CH₂Cl₂. Then 520μL of piperidine (5.26 mmol, 20 equiv.) was added and the solution wasstirred at rt for 1 h. The reaction mixture was quenched by the additionof water and the product was extracted with CH₂Cl₂ (3×), washed withwater (2×), and dried over Na₂SO₄. The LCMS shoved the product and theproduct and piperidine adduct. Next time shorter reaction time and lessequivalents of piperidine should be used. The crude material 102 wassubmitted to next reaction.

A 20 mL vial equipped with magnetic stir bar was charged with 126 mg ofthe amine 102 (0.180 mmol, 1 equiv.) and 0.490 mL of the 24% hydrazinesolution in water (3.67 mmol, 10 equiv.) dissolved in 6.6 mL ofmethanol. After stirring at rt for 12 h, the reaction mixture waspartially concentrated on rotavap (60° C.), quenched by addition of 2MKOH solution, extracted with CH₂Cl₂ (3×) and dried over Na₂SO₄. Theorganics were concentrated and the crude product was purified on silicagel column (30 g) using 0-60% of solvent 2 (solvent 2=30% MeOH inCH₂Cl₂₊₃% NH₄OH) in CH₂Cl₂ as eluent affording 67.0 mg ofN—((S)-3-((R)-3-(((4-aminobutyl)(pyridin-2-ylmethyl)amino)methyl)piperazin-1-yl)-1-phenylpropyl)-4,4-difluorocyclohexane-1-carboxamide103 as a white foam. ¹H NMR (400 MHz, CDCl₃, ppm) δ: 8.54-8.52 (m, 1H),7.97 (d, J=7.0 Hz, 1H), 7.64 (td, J=7.7, 1.8 Hz, 1H), 7.34 (d, J=7.8 Hz,1H), 7.31-7.25 (m, 2H), 7.25-7.15 (m, 3H), 7.15 (dd, J=7.6, 4.9 Hz, 1H),5.04 (q, J=6.1 Hz, 1H), 3.78 (A of AB, J_(AB)=14.7 Hz, 1H), 3.66 (B ofAB, J_(AB)=14.7 Hz, 1H), 3.19 (br s, 3H), 3.06 (dt, J=11.8, 2.8 Hz, 1H),2.91-2.78 (m, 4H), 2.74 (d, J=11.2 Hz, 1H), 2.68 (t, J=6.6 Hz, 2H),2.58-1.39 (m, 23H).

A 100 mL rb flask equipped with a stir bar was charge with 1.92 g of thealdehyde 29 (5.51 mmol, 1 equiv.), 2.14 g of the amine 104 (seesynthesis in ACS Med. Chem. Lett., 2018, 9, 17-22) (6.34 mmol, 1.15equiv.), 1.99 g of NaBH(OAc)₃ (9.37 mmol, 1.7 equiv.) and 22 mL ofCH₂Cl₂ and the suspension was stirred at rt for 12 h. Then the reactionmixture was quenched by addition of sat. NaHCO₃sol., and the product wasextracted with CH₂Cl₂ (3×) and dried over Na₂SO₄. The crude product waspurified on silica gel column (80 g) using 0-60% EA in hexanes as eluentaffording 2.63 g (71%) of 4-benzyl 1-(tert-butyl)(S)-2-((((3,5-dimethylpyridin-2-yl)methyl)(4-(1,3-dioxoisoindolin-2-yl)butyl)amino)methyl)piperazine-1,4-dicarboxylate105 as a yellow oil. Later, when the bottom chain is installed,racemization got identified.

A 100 mL rb flask equipped with a stir bar and rubber septum was chargedwith 2.60 g of the carbamate 105 (3.88 mmol, 1 equiv.), 550 mg of 20 w %of Pd(OH)₂ on carbon (0.780 mmol, 0.2 equiv.), 1.96 g of NH₄OOCH (31.1mmol, 4 equiv.) and 27 mL of dry ethanol. Then the reaction mixture washeated with a heat-gun till bubbling to initiate the catalyst. Afterstirring at rt for 48 h, more 1.96 g of NH₄OOCH (31.1 mmol, 4 equiv.)was added and the reaction mixture was stirred for 24 h. The reactionmixture was filtered through celite plug and the celite plug was washedwith ethanol. The organics were concentrated under vacuum (rotatoryevaporator) and the crude product was dissolved in CH₂Cl₂ and sat.Na₂CO₃ solution was added, the product was extracted with CH₂Cl₂ (3×)and dried over Na₂SO₄. The crude product was purified on silica gelcolumn (40 g) using 0 to 30% Solv2 (Solv2=30% MeOH in CH₂Cl₂ with 3%NH₄OH) in CH₂Cl₂ as eluent affording 1.22 g (59%) of tert-butyl(R)-2-((((3,5-dimethylpyridin-2-yl)methyl)(4-(1,3-dioxoisoindolin-2-yl)butyl)amino)methyl)piperazine-1-carboxylate106 as a yellow oil.

A 100 mL rb flask equipped with a stir bar was charged with 1.11 g of(S)-4,4-difluoro-N-(3-oxo-1-phenylpropyl)cyclohexane-1-carboxamide (3.77mmol, 1.3 equiv.), 1.56 g of the amine 106 (2.90 mmol, 1 equiv.), 33 μLof acetic acid (0.580 mmol, 0.2 equiv.) and 28 mL of DCE. Then 1.05 g ofNaBH(OAc)₃ (4.93 mmol, 1.7 equiv.) was added and the suspension wasstirred at rt for 12 h. The reaction mixture was quenched by addition ofsat. Na₂CO₃ solution, extracted with CH₂Cl₂ (3×) and dried over Na₂SO₄.The crude product was purified on silica gel column (80 g) using 0 to100% EA in hexanes as eluent affording 2.22 g (94%) of tert-butyl(R)-4-((S)-3-(4,4-difluorocyclohexane-1-carboxamido)-3-phenylpropyl)-2-((((3,5-dimethylpyridin-2-yl)methyl)(4-(1,3-dioxoisoindolin-2-yl)butyl)amino)methyl)piperazine-1-carboxylate107 as a white foam. ˜6:1 d.r. was noticed by ¹³C NMR.

A 100 mL rb flask equipped with astir bar was charged with 1.91 g of theamine 107 (2.34 mmol, 1 equiv.) dissolved in 22 mL of CH₂Cl₂. Then 5.42mL of CF₃COOH (70.3 mmol, 30 equiv.) was added. After stirring at rt for5 h, the reaction mixture was cooled in an ice bath and quenched byaddition of 2 N NaOH solution till pH=11, extracted with CH₂Cl₂ (3×) anddried over Na₂SO₄. The crude material was purified on silica gel column(30 g) using 0-30% Solvent 2 (solvent 2=70% CH₂Cl₂, 30% MeOH, 3% NH₄OH)in CH₂Cl₂ as eluent affording 1.51 g (90%) ofN-((1S)-3-(3-((((3,5-dimethylpyridin-2-yl)methyl)(4-(1,3-dioxoisoindolin-2-yl)butyl)amino)methyl)piperazin-1-yl)-1-phenylpropyl)-4,4-difluorocyclohexane-1-carboxamide108 as a yellowish foam.

A 100 mL rb flask equipped with a magnetic stir bar was charged with 251mg of triphosgene (0.840 mmol, 0.410 equiv.) and 20 mL of CH₂Cl₂. Then522 mg of (R)-1-(9H-fluoren-9-yl)ethan-1-ol (2.48 mmol, 1.2 equiv.) wasadded, followed by 200 μL of pyridine (2.48 mmol, 1.2 equiv.) at 0° C.After stirring at rt for 2 h, the reaction mixture was washed with water(3×) and dried over Na₂SO₄. The organics were concentrated and dissolvedin 15 mL dry CH₂Cl₂. Then 1.480 mg of the amine 108 (2.07 mmol, 1equiv.) dissolved in 5.0 mL of CH₂Cl₂ was added at 0° C., followed by458 μL of NEt(iPr)₂ (2.69 mmol, 1.3 equiv.) and the stirring wascontinued at rt for 2 h. The reaction mixture was diluted with CH₂Cl₂,washed with water, brine and dried over Na₂SO₄. The crude material waspurified on silica gel column (120 g) using 0-100% EA in hexanes aseluent affording 1.23 g of (R)-1-(9H-fluoren-9-yl)ethyl(R)-4-((S)-3-(4,4-difluorocyclohexane-1-carboxamido)-3-phenylpropyl)-2-((((3,5-dimethylpyridin-2-yl)methyl)(4-(1,3-dioxoisoindolin-2-yl)butyl)amino)methyl)piperazine-1-carboxylate109 (>99:1 d.r., 62%) as a white foam white foam and 523 mg (3:1 d.r.)of the other fraction of the product.

A 20 mL vial equipped with a magnetic stir bar was charged with 170 mgof the amine 109 (0.184 mmol, 1 equiv.) and 1.8 mL of CH₂Cl₂. Then 55.0μL of piperidine (0.550 mmol, 3 equiv.) was added and the solution wasstirred at rt for 1 h. No reaction was observed. Then 27.8 μL of DBU(0.184 mmol, 1 equiv.) was added and the stirring was continued at rtfor 3h. The reaction mixture was diluted by addition of CH₂Cl₂, washedwith water and brine, and dried over Na₂SO₄. The LCMS shoved the productand the product and piperidine adduct. Next time other nucleophilicamine should be used, like piperazine. The crude material 110 wassubmitted to next reaction.

A 20 mL vial equipped with magnetic stir bar was charged with 188 mg ofthe amine 110 (0.260 mmol, 1 equiv.) and 0.350 mL of the 24% hydrazinesolution in water (2.63 mmol, 10 equiv.) dissolved in 2.6 mL ofmethanol. After stirring at rt for 12 h, more 0.750 mL of the 24%hydrazine (0.520 mmol, 20 equiv.) and 2.6 mL of MeOH was added and thestirring was continued for 12 h. Then the reaction mixture was partiallyconcentrated on rotatory evaporator (60° C.), quenched by addition of 2MKOH solution, extracted with CH₂Cl₂ (3×) and dried over Na₂SO₄. Theorganics were concentrated and the crude product was purified on silicagel column (30 g) using 0-60% of solvent 2 (solvent 2=30% MeOH inCH₂Cl₂₊₃% NH₄OH) in CH₂Cl₂ as eluent affording 113 mg of the product asa white foam. Still minor impurity, the product was repurified on silicagel column (10 g) again affording 93 mg (60%) ofN—((S)-3-((R)-3-(((4-aminobutyl)((3,5-dimethylpyridin-2-yl)methyl)amino)methyl)piperazin-1-yl)-1-phenylpropyl)-4,4-difluorocyclohexane-1-carboxamide111 as a white foam. ¹H NMR (400 MHz, CDCl₃, ppm) δ: 8.16 (d, J=2.1 Hz,1H), 7.94 (d, J=7.1 Hz, 1H), 7.28-7.20 (m, 3H), 7.19-7.13 (m, 3H), 4.98(q, J=6.3 Hz, 1H), 3.70 (A of AB, J_(AB)=16.6 Hz, 5H), 3.68 (br s, 3H),3.58 (B of AB, J_(AB)=13.4 Hz, 1H), 3.00 (d, J=12.0 Hz, 1H), 2.89-1.34(m, 31H), 2.29 (s, 3H), 2.22 (s, 3H).

Biological Assays

HIV-1 and MAGI Tropism assays can be used to establishactivity/cytotoxicity and synergy studies and ADME profiles of compoundsare evaluated using in vitro assays such as CYP2D6/3A4 assays andmetabolic stability in human and mouse liver microsomes (See Tables 2and 3).

Human Liver Microsomes: Pooled mixed gender human liver microsomes at aconcentration of 20 mg/mL were purchased from XenoTech (Kansas City,Kans.). The vials of microsomes were stored at −80° C. and thawed on icebefore each experiment. The microsomes were diluted to 1 mg/mL with 100mM potassium phosphate buffer (pH 7.4).

Mouse Liver Microsomes: Pooled CD-1 mouse liver microsomes at aconcentration of 20 mg/mL were purchased from XenoTech (Kansas City,Kans.). The vials of microsomes were stored at −80° C. and thawed on icebefore each experiment. The microsomes were diluted to 1 mg/mL with 100mM potassium phosphate buffer (pH 7.4).

Experimental Conditions: Test compounds were weighed and dissolved in100% acetonitrile to make 2 mM stock solutions. Verapamil (human, SigmaAldrich) and diphenhydramine (mouse, Sigma Aldrich) served as positivecontrols and were dissolved in 100% acetonitrile to make 2 mM stocksolutions. The 2 mM stock solution of test and control compounds werefurther diluted in sodium phosphate buffer (100 mM, pH 7.4) to 50 μM toensure the acetonitrile content was <0.2%.

The liver microsome assay was prepared in a 1.5 mL Eppendorf tube(Fisher Scientific) with a final volume of 1100 μL. Each reactioncontained sodium phosphate buffer, liver microsomes (1 mg/mL), and testcompound resulting in a final concentration of 3 μM. Following a 5 minpre-incubation of drug and microsomes in a 37° C. shaking incubator, thereaction was initiated with NADPH (110 μL). Aliquots (100 μL) wereremoved in duplicate at 0, 5, 10, 15, and min time intervals andquenched in cold acetonitrile (200 μL). The aliquots were centrifuged at12,000 g for 5 min and the supernatant removed and placed in an LCMSvial. Positive controls were conducted at a final volume of 600 μL togive each time point in a singlet run. A no NADPH negative control withtest and control compound was conducted in singlet (150 μL) at thelongest time point. Controls were processed and analyzed like testcompounds.

LC/MS Analysis

An Agilent Technologies LCMS system consisting of an Agilent 6120quadrupole mass spectrometer equipped with an Agilent 1200 binary pump,degasser, and auto sampler (Santa Clara, Calif.). The analytical columnused for analysis was an XDB-Eclipse Plus C18 column (4.6 mm×150 mmcolumn; Agilent Technologies). The mobile phase for the HPLC was 50 mMammonium formate buffer, pH 3.5 (A) and acetonitrile (B). The sampleswere analyzed using the following 8-minute method: A linear gradientfrom 10% B to 95% B over for 5 minutes, with a 3 min hold at 95%. Anequilibration at 10% B for 5 min was conducted after each run. Sampleinjection volume was 10 μL and the flow rate was 1.0 mL/min. Single ionmonitoring of the mass in positive mode was conducted the molecularweight for each compound. Each time point was assessed on the LCMS andthe area, based on the extracted ion, was manually integrated.

Calculations: The depletion of the compound was monitored as a functionof time, and the area under the curve was manually integrated. Therelative percent remaining was calculated using: Relative %remaining=Area_(Time)/Area_(Time=0)*100. The relative percentages forthe duplicate runs were averaged and the standard deviations werecalculated. Metabolism data averaged duplicates.

Human Liver Microsomes Assay Compound 10 min 30 min 1 58 ± 4.9%  25 ±0.1% 2 65 ± 8.6%  78 ± 2.2% Verapamil 26 ± 1.5% 8.0 ± 3.0%

Mouse, Liver Microsomes Assay Compound 10 min 30 min 1 116 ± 3.1% 93 ±8.9% 2  95 ± 7.2% 96 ± 6.5% Diphenhydramine  64 ± 3.1% 25 ± 4.1%

Recombinant CYP Inhibition Assay: In a drug discovery program, a rapidscreening for cyctochrome P450 (CYP450) inhibitors is a part of theexisting standard for avoiding the development of drugs likely to giveclinical pharmacokinetic drug-drug interactions and associatedtoxicities. A microtiter plate-based, direct fluorometric assay for theactivities of the principal human drug-metabolizing enzymes, CYP2D6 andCYP3A4 can be used and these assays are rapid and compatible withexisting high-throughput assay instrumentation.

Fluorometric Enzyme Inhibition Assays: Test compounds were dissolved in100% organic solvent (CH₃CN or DMSO) to make 30 mM stock solutions.Quinidine (CYP2D6 assay, Sigma Aldrich) and ketoconazole (CYP3A4 assay,Sigma Aldrich) ran as positive controls and were dissolved in 100%acetonitrile to make 1 mM stock solutions. A 100 mM potassium phosphatebuffer was prepared and adjusted to pH 7.4. The 30 mM stock solution oftest and control compounds (1 mM) were further diluted in phosphatebuffer (100 mM, pH 7.4) to ensure the final organic solvent content was<0.2% in the reaction. In a separate falcon tube, a 2× enzyme/substrate(E/S) solution was prepared in phosphate buffer. The final concentrationof CYP2D6 (Corning) and AMMC was 10 nM and 4 μM, and CYP3A4 (Corning)and BFC was 20 nM and 40 μM, respectively. In a separate falcon tube, a2× NADPH regenerating system (NRS) was prepared in phosphate buffer. Thefinal concentration for each component in the assay was as follows:

-   -   CYP2D6 assay=0.008 mM NADPH, 3.3 mM glucose 6-phosphate, 0.4 U        of glucose-6-phosphate dehydrogenase/mL    -   CYP3A4 assay=2.45 mM NADPH, 24.7 mM glucose 6-phosphate, 1.25 U        of glucose-6-phosphate dehydrogenase/mL

Both enzymatic assays were conducted in a 96-well microtiter plate(Black, Corning Costar) with a final volume of 100 μL. Preparation ofthe plate began with the addition of 74 μL of the E/S in the first well,and 50 μL to all subsequent wells (from 2-11). The test compounds (1 μL)were dissolved in the first well to give the first row a final volume of75 μL. A 1:3 serial dilution of the test compound was conducted byremoving 25 μL from the first well and diluting it with the second andso forth until the tenth row. Final concentrations yielded a range from300 μM-0.01 μM. Well 11 contained no enzyme, and well 12 contained noinhibitor. Both were used as controls for background fluorescence. Theplate was incubated for 10 min at 37° C. for CYP2D6, and 30 mins forCYP3A4. After incubation, the reaction was initiated by the addition of50 μL of the 2× NRS to each well.

Immediately (within 1 min) the fluorescence was measured using amicroplate reader (KC4, BioTek). CYP2D6 was monitored at Ex/Em=410/460nm, and CYP3A4 monitored at Ex/Em=410/538 nm in kinetic mode thatscanned every 5 min for 60 mins. Data was exported and corrected forbackground noise by subtracting the blank from the mean value of allother columns. Fluorescence readout was normalized to the fluorescenceintensity of the reaction in the absence of the test substance (well 12,0% inhibition) and the mixture of reaction components in the presence of“inhibitor cocktail” (well 11, 100% inhibition). The percent ofinactivated enzyme for each dilution of test compounds or controls(designated as I %): I=(1−(mean of individual column−mean of column11)/mean of column 12−mean of 11))×100. The IC₅₀ value was derived afterthe data was fitted on a 10-point curve using a four-parameter logisticregression model using Graph Pad Prism 7.

TABLE 2 CYP3A4 and 2D6 IC50 values for the respective compounds.Compound rCYP3A4 (IC₅₀) μM rCYP2D6 (IC₅₀) μM 1 63.46 Range: 43.1 to118.9 58.32 Range: 44.58 to 85.35 2 750.6 Range: >1000 156.2 Range:49.67 to >200

TABLE 3 Activity data for selected compounds. MAGI MAGI MAGI MAGI MAGIMAGI HIV-1_(IIIB) HIV-1_(IIIB) HIV-1_(BaL) HIV-1_(BaL) HIV-1_(IIIB)HIV-1_(BaL) Compound IC₅₀, μM IC₉₀, μM IC₅₀, μM IC₉₀, μM TC₅₀, μM TC₉₀,μM  2 0.07 0.68 2.61 29.1 >10.0 33.1 66 12.4 65.8 0.33 4.38 >100 >100 673.19 44.6 2.33 36.7 >100 >100 68 10.2 70.2 4.16 66.8 >100 >100 77 0.0020.009 1.54 23.0 >100 >100 75 6.61 68.5 2.51 97.1 >100 >100 79 2.58 36.50.66 7.35 >100 >100 81 3.49 52.7 0.95 22.7 >100 >100 URf isomer 95 0.170.90 0.82 8.96 >10.0 >100 LRf isomer 95 >10.0 >10.0 9.36 >100 >10.0 >100

The invention claimed is:
 1. A compound according to Formula (I) or asalt thereof,

wherein R^(A), R^(B), R^(C) and R^(D) are individually and independentlyH, aryl or a C1 to C4 alkyl which may be straight, branched, saturatedor unsaturated, or R^(A) and R^(B), together with the atoms to whichthey are attached, or R^(C) and R^(D), together with the atoms to whichthey are attached, are connected to form a carbocycle, heterocarbocycle,aryl or heteroaryl, and R^(A), R^(B), R^(C) and R^(D) are individuallyand independently optionally substituted with R^(X); wherein R^(E),R^(G1), R^(G2), R^(G3), R^(H1) and R^(H2) are each individually andindependently selected from the group consisting of H, alkyl,carbocycle, heterocarbocycle, aryl and heteroaryl, each of which isoptionally substituted with R^(X); wherein ring W is a carbocycle,heterocarbocycle, aryl or heteroaryl; wherein o is 0, 1, 2, 3 or 4;wherein R^(F) is a chloro, fluoro, bromo, iodo, C1 to C3 alkyl,trifluoromethyl, or O; wherein X is a N or a CH; wherein Y is NH when Zis CO and Y is CO when Z is NH; wherein n, m and p are eachindependently 0, 1 or 2; s is 1, 2, 3, 4 or 5; and wherein R^(X) is ahalogen, nitro, cyano, hydroxy, trifluoromethoxy, trifluoromethyl,amino, formyl, carboxy, carbamoyl, mercapto, sulfamoyl, methyl, ethyl,methoxy, ethoxy, acetyl, acetoxy, methylamino, ethylamino,dimethylamino, diethylamino, N-methyl-N-ethylamino, acetylamino,N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl,N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methylthio, ethylthio,methyl sulfinyl, ethylsulfinyl, mesyl, ethyl sulfonyl, methoxycarbonyl,ethoxycarbonyl, N-methylsulfamoyl, N-ethyl sulfamoyl, N,N-dimethylsulfamoyl, N,N-diethylsulfamoyl, N-methyl-N-ethyl sulfamoyl,carbocyclyl, aryl, or heterocyclyl.
 2. A compound selected from thefollowing structures or a salt thereof:

wherein R^(E) is an optionally substituted alkyl, optionally substitutedcarbocycle, optionally substituted heterocarbocycle, optionallysubstituted aryl, or optionally substituted heteroaryl.
 3. The compoundof claim 2, wherein R^(E) is selected from the group consisting of:


4. A pharmaceutical formulation comprising a compound of claim 1 incombination with a pharmaceutically acceptable excipient, diluent, orcarrier, in the form of a tablet, pill, capsule, gel or aqueous bufferedsolution.
 5. The pharmaceutical formulation of claim 4, furthercomprising an antiviral agent or chemotherapeutic agent.
 6. A method oftreating a viral infection, the method comprising administering acompound of claim 1 to a subject in need thereof, wherein the viralinfection is an HIV infection.
 7. The method of claim 6, wherein thesubject is at risk of, exhibiting symptoms of, or diagnosed with theviral infection.
 8. The compound of claim 2, wherein the compound has astructure of the following or a salt thereof:


9. A pharmaceutical formulation comprising a compound of claim 2 incombination with a pharmaceutically acceptable excipient, diluent, orcarrier, in the form of a tablet, pill, capsule, gel or aqueous bufferedsolution.
 10. The pharmaceutical formulation of claim 9, furthercomprising an antiviral agent or chemotherapeutic agent.
 11. A method oftreating a viral infection, the method comprising administering acompound of claim 2 to a subject in need thereof, wherein the viralinfection is an HIV infection.
 12. The method of claim 11, wherein thesubject is at risk of, exhibiting symptoms of, or diagnosed with theviral infection.